Objective To investigate blood and microbial contamination of surgical shoes of health care workers(HCWs), and provide theoretical basis for the cleaning and disinfection of surgical shoes. Methods From April to May in 2020, 80 pairs of surgical shoes after cleaning and disinfection were randomly selected from the operating room of a hospital for three consecutive weeks, according to shoes used by different staff (surgeons, nurses, anesthesiologists and cleaners), shoes were divided into four categories, blood contamination on instep of shoes was investigated by naked eye observation and occult blood test, specimen of sole taken by contact imprinting method were performed bacterial and fungal culture. Results Occult blood test result showed that 50.00% (40/80) of the surgical shoes were contaminated by blood, contamination rate of instep of surgeons' shoes were the highest, accounting for 63.33%. Bacterial colony count showed that operating shoes of the cleaners were seriously contaminated by microorganisms, bacterial and fungal counts were the highest, which were 93 CFU/cm² and 58 CFU/cm² respectively. Occult blood test and naked eye observation were evaluated, thee results showed that sensitivity and specificity of detection of occult blood by colloidal gold immunochromatography assay were 85.00% and 70.00% respectively. Six shoes were false negative and 12 shoes were false positive. The positive rate of naked eye observation was 57.50%, and that of occult blood test was 50.00%, there was no significant difference between two methods (P>0.05). Conclusion Blood and microbial contamination of HCWs' operating shoes is serious, it is necessary to strengthen cleaning and disinfection, and pay attention to the risk that operating shoes may be the hidden host of pathogenic microbial transmission.