Objective To evaluate the measurement uncertainty for the content determination of Vitamin B12 injection by UV. Methods The model of content determination by UV was established. The uncertainty sources had been analyzed. Each active component of uncertainty was calculated， and the extended uncertainty was obtained. Results The extended uncertainty for the UV determination of Vitamin B12 was 1.0%， the result of content determination was（98. 4±1. 0）%， k=2. Conclusion The main sources of uncertainty were analyzed， which provided a reliable theoretical basis for effectively controlling of this method.