[Background] Rap1 is a kind of small GTPase, and the method of its activity detection is very scanty. At present, the method mainly depends on commercialized kit, resulting in the high cost. RalGDS has the Rap binding domain (RapBD), which can bind to GTP-Rap1 specifically. [Objective] Establish an inexpensive method of detecting human Rap1 activity by exogenous GST-RapBD fusion protein expressed in Escherichia coli. [Methods] We constructed the plasmid with pGEX-4T-1 vector expressing GST-RapBD fusion protein in E. coli, and then combined GST-RapBD with GST affinity resin. Finally, we used GST Pulldown assay to detect Rap1 activity. [Results] The method of detecting human Rap1 activity was established successfully. [Conclusion] Sequence optimization made pGEX-4T-1 highly express the GST tagged RapBD protein in E. coli, which increased the efficiency and decreased the cost of Pulldown assay to detect GTP-Rap1.