Objective To investigate the effect of filtration membrane method and smear method on detection of microorganism in hemodialysis-related water, and provide basis for selecting appropriate detection method. Methods A retrospective study was conducted to analyze the results of water microorganism in hemodialysis-related water detected by filtration membrane method and smear method from January to December, 2018. Bacterial isolation rate, colony forming unit(CFU), rate of intervention-exceeding value as well as qualified rate of microorganism detected by filtration membrane method and smear method were compared and analyzed. Results A total of 274 hemodialysis-related water microbial detection results were collected, including 131 dialysate, 39 reverse osmosis water, 14 concentrated solution B (liquid B), 34 replacement fluid and 56 other dialysis water. Bacterial isolation rates of filtration membrane method were all higher than those of smear method, bacterial isolation rates of dialysate, reverse osmosis water, replacement fluid, and other dialysis water specimens detected by two methods were all significantly different (all P<0.05). The CFU of dialysate, liquid B and other dialysis water specimens detected by filtration member method were all lower than that of smear method, CFU of other dialysis water specimens detected by two methods was significantly different (t=-3.011, P=0.004). Detection rate of intervention-exceeding value of other dialysis water detected by filtration membrane method was lower than that smear method, difference was significant (χ²=6.596,P=0.010). Microbial qualified rate of replacement fluid and reverse osmosis water specimens detected by filtration membrane method was less than that of smear method, qualified rate of microbial detection of replacement fluid was significantly different (χ²=18.987,P<0.001). Conclusion The detection ability of filtration membrane method is higher than that of the smear method, but it has certain limitations in the detection of microorganisms in hemodialysis-related water, except for the replacement fluid, effect of filtration membrane method on the microbial detection of other hemodialysis-related water is no better than that of smear method.