Currently, due to the limitation of hardware for network protocol developing and huge cost of network building in hardware for performance evaluation, most of the literature focuses on software system which limits the results in theory. To solve these problems, a hardware-in-loop simulation system for distributed wireless network MAC (Media Access Control) protocols based on GNU Radio and the second generation of Universal Software Radio Peripheral (USRP2) was designed and implemented. Referring to the standard IEEE802.11 Distribution Coordination Function (DCF) protocol, the designed simulation system adopted the discrete-event simulation technique to realize simulation for multi-node distributed wireless networks with only the least hardware resources (i.e., one Personal Computer (PC) and two USRP2s). In the software, the MAC protocols were implemented using Python language, which is flexible and easy to change or extend. And in the physical layer, modularized modules in C++ language were adopted for signal processing, which further improves the scalability of the simulation system. The experimental results validate the reliability of the hardware-in-loop simulation system, in comparison with the Bianchi algorithm and time slot based saturation throughput calculation model.