This study aimed to validate a tool for assessing vaccine literacy (VL) in adult vaccination under the Chinese context. A pilot study on adult's VL was conducted in 2023 in Huzhou city of Zhejiang province, China. A self-designed questionnaire, specific for the self-assessment of three domains (functional, interactive, and critical), as well as the vaccination knowledge and acceptance was applied. Validation process of the questionnaire was conducted through the investigation of a panel of experts, principal component analysis (factors whose cumulative explanation proportion of variance > 80% were selected) and Cronbach's α coefficient test (>0.7 was acceptable). Correlation coefficient analysis for VL domains, vaccination knowledge and acceptance were also implemented, and r value as well as its significance were calculated. For the validation of the questionnaire, the mean scores for reliability, comprehensibility, efficiency, sensitivity of each question of functional and interactive/critical VL ranged from 3.86 (sensitivity of interactive/critical VL) to 4.26 (comprehensibility of functional VL). Cronbach's α coefficient was 0.8223 with functional VL, 0.8916 for interactive VL and 0.9061 for critical VL. The scores of functional VL, interactive VL and critical VL were 3.36, 3.06 and 2.91, respectively. The mean score of vaccine knowledge was 5.80. The positive response rate of receiving influenza vaccine, pneumococcal vaccine and herpes-zoster vaccine was 36.5% (73/200), 23.00% (46/200) and 11.66% (19/163). The VL score of all domains, the individual's vaccine knowledge and the vaccine acceptance were all significantly correlated to the high education degree (r = 0.37 for functional VL, r = 0.35 for interactive VL, r = 0.30 for critical VL, r = 0.28 for vaccine knowledge, r = 0.42 for vaccine acceptance). The VL questionnaire was shown to be a valid and reliable tool for measuring VL among Chinese adults. It was recommended that the VL questionnaire should be validated in larger and more diverse samples, exploring the impact of interventions to improve VL and assessing the relationship between VL and vaccination behavior over time.