Objective To produce rabbit polyclonal antibodies against human retinol-binding protein (RBP). Methods RBP cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR) and then the amplified products were inserted into prokaryotic expression vector pET-28a(+) to construct recombinant plasmid pET-28a(+)-RBP. The established plasmid was then transformed into E. coli. Isopropylthio-β-D-thiogalactoside (IPTG) was used to induce the expression of recombinant protein His-RBP in E. coli. The expression products were identified by SDS-PAGE from different clones of E. coli to screen positive bacteria, followed by amplifying culture. His-RBP protein was purified from the expression products of positive clones. The purified recombinant His-RBP was used to immunize New Zealand white rabbits. Antisera were acquired after four times of booster immunization. The prepared purified polyclonal antibodies were identified by SDS-PAGE, ELISA and Western blotting. Results We successfully constructed the recombinant plasmid pET-28a(+)-RBP, and acquired recombinant protein His-RBP of high purity. ELISA showed that the antibody titer reached 1:512 000. Conclusion The rabbit polyclonal antibodies against human RBP have been successfully prepared.