Heat release rate is a critical parameter in the combustion process, yet its quantification can be challenging to obtain experimentally. In this study, an acoustical measurement system based on the Rijke tube was established to investigate the heat release rate perturbations of 25 propane/air flames under different mixing conditions. For premixed flames, perturbations in CH* and OH* chemiluminescence were consistent with the heat release rate disturbances determined using the acoustical method, thereby validating the proposed acoustical method and platform. Based on these, a comprehensive analysis was carried out on the chemiluminescence results and heat release rate perturbations of flames under different fuel–air mixing conditions, revealing the condition where the chemiluminescence method no longer reliably indicates the heat release rate. The research also investigated the chemiluminescence structure of flames under different fuel–air mixing conditions, identifying a difference in the distribution of CH* and OH* chemiluminescence at higher equivalence ratios (approaching diffusion flame conditions). In fully diffusion flames, the perturbation of OH* chemiluminescence was primarily observed near the base of the flame, while the peak perturbation of CH* chemiluminescence resides within the soot reaction zone downstream of the flame front.