Peroxynitrous acid (ONOOH) produced by online mixing of nitrite and acidified hydrogen peroxide could induce weak chemiluminescence (CL). A stronger chemiluminescence was observed in the presence of gold nanoclusters (Au NCs). This novel CL system was developed as a flow-injection method for the nitrite determination directly and conveniently. This method was achieved in acidic medium, which greatly improved its selectivity. The CL mechanism of the peroxynitrous acid–gold nanocluster system was investigated using CL spectroscopy, UV-visible spectroscopy and radical scavengers. The enhanced CL could be attributed to the catalysis of Au nanoclusters. The proposed method has been applied to determine nitrite in water samples with good accuracy and precision.