Periodontal disease is an inflammatory process in the periodontal tissue that is initiated by the microorganisms in the subgingival plaque. If left untreated, it will cause periodontal tissue breakdown and alveolar bone resorption. Porphyromonas gingivalis （Pg）、Tannerella forsythia （Tf） and Treponema denticola （Td） are among the anaerobic species frequently associated with periodontal disease, which possess a trypsin-like enzyme that can be detected by the hydrolysis of N-benzoyl-DL-arginine-2-naphthylamide （BANA）. The purpose of the study is using the BANA test to detect the presence of trypsin-like enzyme releasing periodontopathogenic bacteria from patients. For each qualified periodontal patients, two samples were taken from advanced lesion （pocket depth （PD） > 5 mm）, two from mild lesion （PD < 5 mm）, and one was saliva sample. Endodontic paper points were inserted into the periodontal pockets to collect microbiological samples. One hundred μl Wilkins Chalgren medium and 100 μl BANA solution then were incubated with each sample overnight at 37 °C. The color was developed by the addition of 10 μl fast garnet solution and read with an enzyme linked immunosorbent assay reader with a 490 nm filter within 30 min. The data showed that the optical density values for samples collected from advanced lesions were significantly higher than those from less serious lesions. The results suggest that the advanced periodontal lesion may harbor higher numbers of periodontal pathogenic bacteria （ie, red complex）, which produce high titers of trypsin-like enzyme in gingival fluid and can be detected by BANA test. The BANA test may useful in quick detecting the severity and activity of periodontal pathogenic bacteria in patients.