ObjectiveTo establish a reliable approach for quantification of colony forming unit(CFU) of Mycobacterium tuberculosis(M.tb) by measuring optical density(OD).MethodsM.tb suspension H37Ra was prepared using lowpower ultrasonic or glass bead beating methods, and was twofold serially diluted, OD at 600nm (OD600) of each dilution ratio was measured respectively, OD600 and dilution curve were analyzed to determine the optimum approach for preparing bacterial suspension，linear range of OD600, as well as linear relationship between OD600 and CFU.ResultsOD600 was 0.1-0.6, linear regression analysis of OD600 and dilution ratio within linear range revealed that correlation coefficient (R2) of glass bead beating and lowpower ultrasonic methods were 0.98 and 1.00 respectively，both presented a good correlation, lowpower ultrasonic method was better than glass bead beating method, bacterial suspension dispersed more evenly. Linear regression analysis results of OD600 and CFU values showed that the regression equation of glass bead beating method and lowpower ultrasonic method were CFU=2.35×107×OD600+4.42×105 and CFU=3.26×107×OD600+6.89×105 respectively.ConclusionLowpower ultrasonic method is a good method for preparation of M.tb suspension，combined the measurement of OD600 value， it can be a reliable and rapid method for quantitative analysis of M.tb.