[Objective] To explore the effect of disclosing agent methylene blue (MB) on tissue samples bacterial culture in periprosthetic joint infection (PJI). [Methods] Based on the modified diagnostic criteria of the Muscular Skeletal Infection Society (MSIS), a total of 116 patients with PJI who were treated with two-stage revision surgery of hip or knee from October 2017 to October 2019 were included in the study. At least 3 periprosthetic tissues were taken out during surgery for bacterial culture. Each tissue was divided into two equal parts. One of the tissues was stained with 0.1% MB solution and the other was used as a control group without staining. Compare the final positive rate of culture and results of bacterial identification between the two groups. [Results] The positive rate of tissue culture in two groups was 81.9% and 86.2% in 116 patients, respectively, with no significant difference between them (P=0.370). The tissue culture results in the two groups showed MB has some antimicrobial effects on Klebsiella oxytoca and Coagulase-negative Staphylococcus other than Staphylococcus haemolyticus. [Conclusion] MB solution may affect the detection of a certain bacteria in PJI.