Abstract: A HPLC method was introduced to determine the content of Vitamin B₁₂ in 47 batches of gentamicin sulfate, procaine hydrochloride and Vitamin B₁₂ capsules from 4 manufacturers including factory A, factory B, factory C, factory D, and the content uniformity was evaluated for Vitamin B₁₂ in the same batch or different bathes of samples from 4 manufactures. Vitamin B₁₂ in capsules was extracted with water. CAPCELL PAK ACR C₁₈ chromatographic column was used as separation column, the mixtures of methanol and 0.028 mol·L^-1 sodium dihydrogen phosphate solution (pH 3.2) with various volume ratios were used as mobile phase for gradient elution, and the content of Vitamin B₁₂ was determined at the detection wavelength of 362 nm. The results showed that the linearity range of Vitamin B₁₂ standard curve was in the range of 0.2-5.0 mg·L^-1, with lower limit of determination (10S/N) of 0.12 mg·L^-1. The spiked recovery was made on capsule samples at three concentration levels, giving recoveries in the range of 96.9%-102%, and RSD of measured values of Vitamin B₁₂ in 6 sample solutions was 1.5%. This method was used to analyze the 47 batches of samples, and values of Vitamin B₁₂ obtained were in the range of 55.4%-137.0% of the labelled values of the samples from factory D, exceeding the range (80.0%-120.0%) set by the second part of Chinese Pharmacopoeia and actual samples. Therefore, the content uniformity evaluation was made, and the results showed that the vitomin B₁₂ content in the samples within and between batches was uneven, finding that it may be related to the dry mixing process used in factory D.