Abstract: A method based on microscopic imaging flow cytometry was established to measure the concentration of milk somatic cells in reference materials for milk somatic cell count. This method labels milk somatic cells using the fluorescent dye ethidium bromide. The single-cell multi-parameter microscopic images provided by microscopic imaging flow cytometry can effectively assist in identifying target cells in flow cytometric analysis, thereby improving the accuracy and repeatability of gating strategies. Through multiple measurements and analyses of milk somatic cell reference material samples with different somatic cell concentrations, it has been proven that this method has good intra-laboratory and inter-day repeatability. A mathematical model for measuring the concentration of milk somatic cells in reference materials for milk somatic cell count based on microscopic imaging flow cytometry was established. The analysis determined that the main sources of uncertainty in this measurement method come from measurement repeatability, sample pretreatment, reagent addition, gating operation, and instrument measurement volume deviation. Comprehensive evaluation targeting the candidate Grade V (～1.0×10⁶ cells/mL) reference material for milk somatic cell count prepared in this study shows that the relative uncertainty of the measurement method for milk somatic cell concentration based on microscopic imaging flow cytometry is 4.84%.