Background: Preconception care is the provision of biomedical, behavioral and social health interventions to women before conception occurs to improve their health status and pregnancy outcome. Despite its importance in promoting maternal and child health, women’s preconception care knowledge, the use of this service and its determinants are not well investigated in Ethiopia. Objective: This study aimed to assess knowledge, practice and associated factors of preconception care among pregnant women attending antenatal care at public health facilities in Injibara town, Northwest Ethiopia, 2020. Materials and Methods: An institution based cross-sectional study was conducted among 401 pregnant women from December 4/2020 to January 2/2021 in public health facilities of Injibara town. The Study subjects were selected by systematic random sampling technique. The data were collected by face-to-face interview using a structured questionnaire. The data was entered into Epi- data version 4.6 and analyzed using SPSS version 20. Both descriptive and analytical analysis was computed. Bivariate and multivariable binary logistic regression analysis was used to identify factors associated with the knowledge and practice of preconception care. Statistical significance was considered at P-value <0.05. Model fitness was checked using Hosmer Lemeshow test with p-value >0.05. Results: Among the study participants 69(17.2%) and 49(12.2%) of pregnant women had good knowledge and good practice towards preconception care respectively. This study identifies that having pre-existing medical illness (AOR=3.36, 95% CI: 1.44-7.82), intended pregnancy (AOR=2.53, 95%CI: 1.07-5.94), government employment (AOR=7.15, 95% CI: 3.29-15.5), husband support (AOR= 4.22, 95%CI: 2.10-8.48) and family planning use (AOR=2.30, 95%CI: 1.10-4.81) were significantly associated with women’s knowledge of preconception care. Knowledge on preconception care service (AOR=11.83, 95% CI: 5.06-27.6), adverse pregnancy outcomes (AOR=4.16, 95%CI: 1.65-10.48) and husband support (AOR=5.08; 95% CI: 1.93-13.38) had a statistical association with preconception care practice. Conclusion: In these study women’s level of knowledge and practice towards preconception care were very low. Knowledge on preconception care service, husband support and adverse pregnancy outcomes were affect preconception care practice positively. Increasing women’s knowledge regarding preconception care and promoting husband support is important. Key words: Barrier, preconception care, knowledge, practice, Injibara town.