The invention discloses a chromatographic purification method for acylated insulin and belongs to the field of preparation of recombinant insulin or insulin analogs. According to the chromatographic purification method for acylated insulin, high-grade silica gel carriers (like C4, C8 and C18 alkanes taken as reversed phase filler of ligands) are taken as chromatographic filler, a high-pressure chromatographic system is used for performing fine separation and purification on acylated insulin, the purity of a coarse product can be improved from 50%-60% to 99.0% or higher in the alkaline eluent environment with pH (potential of hydrogen) being 6.5-8.0, and the yield of the purified sample can be higher than 90%. Besides, according to the method, the loading quantity of coarse samples is larger, and the purification cost is greatly saved; the linear flow speed is high in the chromatographic purification process, and the insulin analogs can be rapidly purified. The chromatographic purification method is simple, stable and reliable to operate, has higher amplification and is suitable for industrial preparation of the insulin analogs.