Objective Giardia lamblia is an important pathogen of zoonosis giardiasis, it poses a potential threat to the quality of SPF (specific pathogen-free) laboratory animals cannot be ignored. The aim of this study is to establish the method of rapid diagnosis of Giardia lamblia, and analyze the test results of 516 batches form 17 manufactures. Methods Direct microscopy, Giemsa-fast staining and multiplex polymerase chain reaction (multiplex PCR) were applied to detect Giardia lamblia. Results Numerous of Giardia lamblia trophozoites and cysts were detected in SPF laboratory animals by using direct microscopy and Giemsa-fast staining, and multiplex PCR were performed to identify 18S rDNA, β-giardin, TPI and GDH genes of DNA extracted from these trophozoites and cysts identified Giardia lamblia. Direct microscopy, Giemsa-fast staining, and multiplex PCR methods can be used to detect Giardia lamblia. Of the 2562 SPF laboratory animals studied, 22.9% (586/2562) were positive for Giardia lamblia. Conclusions Direct microscopy, Giemsa-fast staining, and multiplex PCR were effective techniques with high sensitivity and specificity for rapid diagnosis of Giardia lambliain. It is not satisfactory that the results of Giardia lamblia examination in 516 batches form 17 manufactures failed to meet the requirements 100%.