Homogentisic acid gamma-lactone (HAL) chemiluminescence (CL) was applied to the determination of horseradish peroxidase (HRP) encapsulated in liposomes. HRP was detected after the lysis of HRP-trapped liposomes with Triton X-100. CL response rate, detection limit and linear range of calibration curve for HRP in HAL CL were compared with those in piodophenol (p-IP)-enhanced luminol CL. Maximal light emission in HAL CL appeared more rapidly compared to that in p-IP enhanced luminol CL, thus resulting in remarkable reduction of CL measurement time. The detection limit for HRP in HAL CL was the same as that in p-IP-enhanced luminol CL. The linear range of calibration curve for HRP in HAL CL was improved by a factor of 50 compared with that in p-IP-enhanced luminol CL. From these results, it was found that HAL CL were superior to p-IP-enhanced luminol CL for the determination of HRP encapsulated in liposomes.