Phages are abundant and ubiquitous in nature, and are therefore important components of microbial communities. The investigation on the survival environment of bacteriophage in the nature might lay a foundation for the further study on regulating function of phage in the microecological environment. To understand the ecology of mycobacteriophage, we collected different types of soil samples from three areas in China to determine the isolation rates of phages and analyzed the relationship between isolation rates and four affecting factors in this study. The mycobacteriophages were isolated from 50 soil samples with mixed lyosol of citric acid and disodium hydrogen phosphate dodecahydrate as extractant. The isolation result was detected by the double agar method and dipping method. At the same time, pH value, moisture, organic carbon level and cation exchange capacity of samples were measured. Thirteen strains of mycobacteriophages were isolated and named from CJAUS6 to CJAUS18. The morphological observation under the transmission electron microscope showed that, all the isolates have a regular icosohedron head [(86.5±5.83-108±23.2)nm ×(83.2±3.04-93.2±5.92)nm ] and a contract tail [(73.0±8.36-87.7±1.86)nm ×(19.3±1.44-30.7±8.78)nm]. So they can be classified as a member of the order Caudovirales and the family Myoviridae. The isolation results from five types of soils indicated that the isolation rate (41.2%) from dark brown earth was highest, followed by yellow brown earth (25.0%) and drab soil (16.7%), while no phages were isolated from the heilu soil and brown earth samples. The reason might relate to three factors as follows: firstly, the organic carbon level is the most in the dark brown earth; secondly, the influence of weathering and eluviatin on dark brown soil is weaker than that on yellow brown earth and drab soil; thirdly, the dark brown earth in this paper was affected by microclimate. In order to know the relationship between the isolation rate and affecting factors, the two-dimentional coordinates graphs were drawn by means of Microsoft Office Excel Software. The result showed that the isolation rate was the highest when the pH value was between 7.45 and 7.95, and moisture was between 13.7% and 21.7%, and the curve of their relationship was expressed as a same inverted "V" form. When the cation exchange capacity was between 20.8cmol/kg and 28.6cmol/kg, the isolation rate had a positive proportion with cation exchange capacity. It is supposed that the way in which cation exchange capacity affects isolation rate might attribute to the change of concentration and composition of anions and cations in the soil solution. And we found the concentration and composition of anions and cations is proportional to the adsorption quantity of phages. There was no obvious correlation between the isolating rate and organic carbon capacity. The results indicated that the soils types and physical and chemical properties of soils might contribute to the isolation rates of mycobacteriophage. Further work will be performed on the relationships between bacteriophage pI and soil pH values, and between water content in soil and the titres of host bacteria.