Abstract: Disclosed is a method for preparing a precursor of a recombinant human insulin or an analogue thereof, comprising: a. bacterial fermentation, centrifuging a fermentation broth in a continuous flow to collect a supernatant; b. filtering the supernatant in step (a) by means of a hollow fiber membrane and collecting the filtrate; and c. purifying the filtrate in the step (b) by means of a chromatography column. The method has the advantages of streamlined steps, scalability, no use of organic solvents, high yield, etc. The purity of the insulin precursor can exceed 90%, the host cell protein removal rate exceeds 90%, and the exogenous DNA removal rate is 89% or greater, achieving less than 0.1 ng/mg.