Fluoresceinyl cypridina luciferin analog (FCLA) is a chemiluminescence (CL) probe for detecting reactive oxygen species (ROS). Its efficiency for detecting singlet oxygen (¹O₂) can be significantly enhanced in the presence of human serum albumin (HSA). This phenomenon may apply to important applications for both research and clinical testing, because of the broad presence of HSA in the human system. In the current study the mechanism of the FCLA–HSA CL system is studied by means of direct CL measurement and spectroscopy techniques. Our results show that FCLA can combine with HSA via a single binding site to form a complex. The CL efficiency of the system is largely governed by an intersystem energy transfer between the two components upon interaction with ¹O₂. The CL production reaches maximum in a synergetic manner when equal amounts of FCLA and HSA are present simultaneously, but production is less at other ratios. The results also show that the combination of FCLA with HSA does not significantly alter the ROS selectivity of FCLA. In conclusion, our study shows that FCLA and HSA can combine and form a complex with higher CL efficiency. This provides us a new approach in designing CL techniques for studying ROS.