Objective To develop a 16S rRNA gene clone library to detect bacterial flora in fecal specimen.Methods DNA of bacteria was extracted from clinical specimen, and general-primer PCR reaction, cloning and sequencing were performed. The nucleotide sequences of bacteria were compared with those in GenBank database.Results The analysis of 16S rRNA gene clone library showed that only 4 kinds of defined bacteria were detected and bacteroides fragilis was 92% in the diarrhoeal fecal specimen, but in the relieved patients the flora distribution inhibited diversity and 12 kinds of defined bacteria were detected, among them bacteroides fragilis was 14.2 %.Conclusion The cultivation-independent bacterial identification system based on 16S rRNA gene sequence analysis is a useful method for analysis of bacterial distribution and can be directly applied in detection of clinical specimen.