Difflugia is a morphologically diverse genus of the free-living shelled amoeboid protozoa that are important components of freshwater ecosystems and play crucial roles in nutrient cycles and energy flow through food webs. Difflugia tuberspinifera is an endemic species of East Asia and is widely distributed in freshwater lakes and reservoirs in China. Clearly, this species plays a critical role in maintaining the structure and function of freshwater ecosystems. However, little is known about its diet composition and predatory behavior at both species and gene levels. This testate amoeba (D. tuberspinifera) was first found in the Wujiang River, Guizhou Province, China. Subsequently, more detailed studies on its morphology and biometry have been done on natural populations from Yangtze River and Pearl River valleys, and Fujian reservoirs. Recently, Han et al. illustrated that D. tuberspinifera is an active and agile hunting carnivore that can capture swimming prey including micro-particulates, rotifers, and other metazoans. In previous studies, however, the specimen identifications were done by microscopic examination, which requires a broad and deep taxonomic knowledge and is very laborious, time-consuming, and somewhat variable because of insufficient taxonomic resolution. In order to facilitate the identification of diet composition in D. tuberspinifera, an inexpensive, efficient, rapid and easy-to handle gene clone library has been developed for diet detection and analysis. The plankton samples were collected from the Hubian Reservoir in Xiamen city in September of 2010. Individual cells were isolated using a glass capillary under an inverted microscope and washed 3-5 times with distilled water before DNA extraction and PCR amplification. The 18S rRNA gene was amplified by the universal eukaryotic primers, and the purified PCR products were ligated into the pGEM-T vector and transformed into Escherichia coli DH5α-competent cells. In total, 46 plasmids containing target gene fragments were successfully identified and sequenced. Each sequence was compared with sequences available in the GenBank database using BLAST, and the closest relatives were identified for diet or food composition analysis. Finally, 11 OTUs (operational taxonomic units) were identified at 97% sequence similarity level, and they belonged to either Rotifera (52%) or Copepoda (48%). Our results, combined with existing data, suggested that: 1) the diet composition of Difflugia tuberspinifera is composed of both rotifera and copepoda species; 2) unicellular protozoa are not only the food of metazoa, but they can also prey on multicellular micro-metazoa; 3) molecular methods are universally applicable, and the SSU rRNA (small subunit ribosomal RNA) gene clone library is an efficient, rapid, and repeatable approach to study the diet composition of protozoa.