A sensitive and selective method for the determination of procaine hydrochloride with a Nafion-modified glassy carbon electrode has been developed. The voltammetric behavior of procaine hydrochloride on the Nafion-modified electrode indicated that the modified electrode not only increased the sensitivity of the determination of procaine hydrochloride, but also catalyzed the electrode process. Procaine hydrochloride was accumulated in Britton-Robinson buffer (pH 2.09) at a potential of -0.2 V (vs. SCE) for 180 s, and was then determined by differential pulse adsorptive stripping voltammetry. The effect of various parameters, such as the pH of the medium, the mass of drop-coated Nafion, the accumulation potential, the accumulation time and the scan rate, were investigated. Under the optimum conditions, a linear calibration graph was obtained in the concentration range of 6.0 × 10-8 to 6.0 × 10-6 mol l-1 with a correlation coefficient of 0.9987. The relative standard deviation was 4.18% for eight successive determinations of 1.0 × 10-7 mol l-1 procaine hydrochloride, and the detection limit (three times signal to noise) was 7.0 × 10-9 mol l-1. A study of interfering substances was also performed, and the method was applied to the direct determinations of procaine hydrochloride in the injection solution of procaine hydrochloride and in rabbit serum.