The present disclosure discloses a reagent for differentiating and counting leukocytes which includes: (1) cationic cyanine compounds selected from those having the following general formulae I and II; (2) cationic surfactants selected from those having the following general formulae III, IV and/or V; (3) at least one nonionic surfactant; and (4) optionally, at least one anionic compound selected from those having one or more carboxyl or sulphonyl groups; and optionally includes alcohol compounds. Also disclosed is a kit comprising the reagent for differentiating and counting leukocytes. Further disclosed is a method for differentiating and counting leukocytes using the reagent and kit. Use of the reagent, kit and method disclosed enables the identification of leukocytes in blood samples into five subtypes in a very short time, or at least achieves the differentiation and counting of four leukocyte groupings. Moreover, immature and abnormal cells can be identified.