Background The human papillomavirus vaccine is one of the main preventative measures for cervical cancer. However, global vaccine uptake is low; the problem is particularly acute in low and middle-income countries. The purpose of this study is to assess female preparatory school students’ knowledge, attitude, and uptake of the human papillomavirus vaccine and associated factors in Bahir Dar City, Ethiopia. Methods Institutional-based cross-sectional study was conducted among 633 female preparatory school students in Bahir Dar city from March 1–30, 2021. Participants were selected using the multistage sampling technique. Data were collected using a structured self-administered questionnaire and entered into Epi-data and exported to SPSS for analysis. Binary and multivariable logistic regression analyses were done using an odds ratio with a 95% confidence interval. Finally-value < 0.05 was considered significant in multivariable analysis. Result The proportion of Human Papillomavirus (HPV) vaccine uptake, knowledge of the vaccine, and respondents’ attitudes toward the vaccine were 45.3% (95% CI = 41.6–49.4%), 58.1% (95% CI = 54.4–61.9%), and 16% (95% CI = 13.2–19.5%), respectively. Having a history of sexual contact AOR = 2.80 (95% CI = 1.64–4.76), hearing about HPV infection AOR = 1.59 (95% CI = 1.13–2.24), and having a positive attitude toward HPV vaccine AOR = 1.46 (95% CI = 1.03–2.08) were significantly associated with knowledge about the HPV vaccine. Discussion of reproductive health issues with family AOR = 2.558 (95%CI = 1.800–3.636), and having good knowledge about HPV vaccine AOR = 3.571(95%CI = 2.494–5.113) were associated with a positive attitude toward the HPV vaccine. Good knowledge AOR = 2.36(95%CI = 1.48–3.76) and a positive attitude toward HPV vaccine AOR = 2.87(95%CI = 1.70–4.85) were strongly associated with HPV vaccine utilization. Conclusion In this study, there was a very low uptake of HPV vaccination among female students, and only a small proportion of them had good knowledge of the HPV vaccine and a favorable attitude toward the HPV vaccine.