Anaerobic ammonium-oxidation (anammox) with nitrite as an electron acceptor has been discovered as both a completely different pathway of the nitrogen cycle and a new cost-effective method for removal of ammonium from wastewater. Recently, the study of the peculiar biochemical mechanism, special structure and function of anammoxosome, functional enzymes and related genes, microbial ecological biotope, and diversity of the anaerobic ammonium oxidation have obtained great improvement, which provided theoretical foundation for the application of anammox bacteria. Especially in the wastewater treatment engineering, the anammox process has become the hotspot in research of new biological N-removal technique for wastewater treatment with its advantages of abbreviation on the N-removal process and low material and energy consumption. Deeper understanding in their species diversity, distribution in the sludge, and qualitative and quantitive relationships with other bacterial species will significantly accelerate the renovation of N-removal technique for wastewater treatment. A summary on the advances in processes on the main physiology characteristics, structural characteristics, biochemical mechanism, applications in the wastewater systems and species analysis of anammox in the engineering process by molecular biological methods are all presented in this paper. Firstly, the anammox reaction takes place inside the anammoxosome: an intracytoplasmic compartment bounded by a single ladderane lipid-containing membrane, and the ladderane membrane lipids have so far been found only in the anammox bacteria. Secondly, all the anammox bacteria are affiliated within 85% sequence similarity of 16S rRNA genes in the branch of Planctomycetes and different levels of the anammox bacterial diversity and distribution are found in various ecosystems. Thirdly, the nitrite reductase, hydrazine hydrolase, and hydrazine oxidizing enzyme are the essentially functional enzymes. Fourthly, the discovery of anammox bacteria and its anaerobic metabolism of ammonia oxidizers open up new possibilities for nitrogen removal from wastewater, which made the paradigm that the only way to biologically convert wastewater ammonium to dinitrogen gas necessitates the complete oxidation to nitrate followed by heterotrophic denitrication become obsolete. Finally, the implementation of the molecular biological methods in the ANAMMOX-related wastewater treatment systems has broadened our information towards the anammox bacteria. The research hotspots of anammox bacteria in both biochemical characteristics and application in wastewater treatment systems are also summarized here. On the aspect of biochemical characteristics, the issues addressed are: (1) some new metabolic reactions besides the anammox reaction have been found, while their theories and reasons were still unclear; (2) the distribution inclination of anammox bacteria in different ecosystems seems to provide some rule which is still unidentified; (3) new kinds of anammox bacteria wait for discovery to expend the pholygenetic relationship, all of which would increase the genetic and ecological spectrum of anammox bacteria in the nature. In terms of the wastewater treatment application, the following issues are dealt with: (1) proper designs and methods for higher efficiency in the enrichment and stabilization of anammox bacteria are required in order to shorten the start-up process of the ANAMMOX-like wastewater treatment plants; (2) more primers or probes with high specificity to anammox bacteria are also needed to guarantee the precision and credibility of research study; (3) the coexistence relationship of anammox and other groups of bacteria, which are still not confirmed, would facilitate the exploration of new anammox metabolism and characteristics, all of which would advance the actual application of anammox bacteria in the wastewater treatment plants.