Using 4-nitro phthalic acid as starting material, two phthalate ester haptens dibutyl 4-aminophthalate (1a) and di(hexan-3-yl) 4-aminophthalate (1b) were synthesized by the esterification with n-butyl alcohol and 2-ethylhexanol, respectively, and then via iron powder reduction. Then hapten 5-((3,4-bis(((2-ethylhexyl)oxy)carbonyl)phenyl)amino)-5-oxopentanoic acid (1c) was prepared by the amidation of hapten 1b and dihydro-2H-pyran-2,6(3H)-dione. Their structures were characterized by melting point, 1H NMR and MS. Then haptens 1a-1c were coupled with bovine serum albumin (BSA) and ovi albumin (OVA), respectively, and six antigens 2a-2c and 3a-3c were prepared and analyzed by ultraviolet absorption spectrum.