The invention discloses a cyclosporine conjugate and a preparation method and application thereof. The cyclosporine conjugate is as shown in general formula (I), wherein n is the molecular number of cyclosporine combined with one bovine serum albumin molecule and is an integer ranging from 1 to 20, BSA is bovine serum albumin, and the molecular weight range is 6.6-6.9KDa. The theophylline conjugate is formed by coupling cyclosporine hapten with carrier substance bovine serum albumin or ovalbumin generating immunogenicity. The preparation method includes: connecting and combining cyclosporine with the carrier substance generating immunogenicity to form the cyclosporine conjugate capable of inducing animal systems to generate antibodies. The cyclosporine conjugate immunizes New Zealand whiterabbits to prepare antiserum with the titer reaching 50,000, and the lowest detection limit of the antiserum is 20ppb. The cyclosporine conjugate is simple in method, fast, accurate and high in specificity and lays a foundation for the enzyme linked immunosorbent assay kit of cyclosporine.