Sixteen kinds of human immunodeficiency virus (HIV) target genes were cloned by polymerase chain reaction (PCR) amplification, and specific plasmids were constructed as the templates for the expression of these genes in the cell-free system. Similarly, the linear PCR templates of these genes for cell-free protein expression were also constructed by using two PCR amplification process. These different templates can be employed to biosynthesize HIV proteins in the cell-free system simultaneously and can be adapted for some high-throughput processes. HIV protease (P10) was performed as a target protein, and two different templates (plasmid and PCR product) were prepared and used for P10 expression in the Escherichia coli cell-free system. The target protein P10 was detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels either by using a plasmid template or by a PCR template. These results are promising and helpful to develop a high throughput process for drug discovery.