Belonging to the technical field of fluorescence in-situ hybridization detection, the invention relates to an absolute quantification method for fluorescence in-situ hybrid cell counting. The absolute quantification method mentioned in the invention adds a weight quantification step in a fluorescence in-situ hybridization process, makes improvements to sampling, sample pretreatment, experimental treatment, late-stage result analysis and other stages of an original experimental scheme, so that the complete set of method is established. The method is characterized in that: when quantifying a biological membrane and other solid samples, a quantitative high speed centrifuged sample can be adopted, high temperature drying is performed on the same sample at the same time, then the sample is subjected to fluorescence in situ hybridization and observation under a microscope as well as calculation, thus finally obtaining the microorganism concentration of dry sludge with certain weight. The method realizes comparison of different forms of samples in a same reactor in terms of microbial community numbers, and at the same time, puts forward an absolute quantification means able to establish a standard method.