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Introduction Confidence in vaccine safety and effectiveness is an important predictor of vaccine uptake. This study assessed the level of knowledge of vaccination and factors associated with vaccination uptake in 28 EU countries. Methods A secondary dataset analysis was performed on data from the Eurobarometer on Europeans’ Attitudes Towards Vaccination (March 2019 across 28 EU countries). Knowledge about vaccination was assessed with the Eurobarometer with four true/false questions (range 0–4 correct answers), with high knowledge reported as a score of 3/4 or 4/4. Adjusted regression analyses were performed on all respondents (n=27524) and those who lived with a child aged 0–14 years (n=6005). Results Significant inter-country variability in the knowledge of vaccination was noted across the 28 European Countries, with little intra-country variation by gender and age. Adults who trusted health authorities as a source of information were more likely to have high knowledge about vaccination (AOR=1.29). In contrast, those who reported the internet as a source of information were less likely to have high knowledge (AOR=0.71). Previous vaccination in the past five years increased with a higher knowledge score among adults. Similarly, the odds of previous vaccination for their children were significantly higher among those who scored 3/4 (AOR=2.17; 95% CI: 1.41–3.34) and those who scored 4/4 (AOR=3.15; 95% CI: 2.01–4.91) than those who answered incorrectly to all questions. Conclusions Higher socioeconomic status, higher educational level, previous vaccination and higher perceived effectiveness of vaccination were significantly associated with higher knowledge of vaccinations. Improving awareness of the benefits of vaccines is warranted, especially in light of COVID-19 booster vaccinations in Europe.