This paper takes mixed algae as the research object to study the influence of single cell variable fluorescence (SCVF) differences of algal species in mixed samples on the accuracy of variable fluorescence statistical analysis. The research results show that when the SCVF ratio of two algae is no more than 5.3, the analysis is still applicable, and the mean absolute value of the relative errors between measurement results and microscopic examination results is no more than 17%. When the SCVF ratio of two algae is higher than 8, the measurement results are quite different from the microscopic examination results. In addition, when high SCVF algal cells account for a large proportion, the measurement results of variable fluorescence statistical analysis only reflect the number of high SCVF algal cells in the mixed samples. As a result, for the mixed algae with large differences in SCVF, the paper combines a filtration separation method with the variable fluorescence statistical analysis method. The mixed samples are filtered by a metal filter screen with suitable pore size, and the density sum of measured viable algal cells before and after filtration is taken as experimental result. The results show that the proposed method can greatly reduce the mean absolute value of the relative error from 58.4% to 5.5%, effectively mitigate the influence of the SCVF difference of algal species in mixed samples on the quantitative analysis of cell number, and accurately quantify viable cells in mixed algae.