Background Breast self-examination (BSE) is a simple, low-cost, and non-invasive method that women use to detect breast cancer at early stages. BSE is recommended for teenage and adult women as it helps to detect unusual swelling and lumps. The early intervention reduces the spread of cancer cells and improves health outcomes. This study assessed the knowledge and practices of BSE for early detection of breast cancer among women attending maternal health clinic at Jaramogi Oginga Teaching and Referral Hospital (JOOTRH), Kisumu County in Kenya. Methods A cross-sectional study targeting women of reproductive age seeking maternal health services at the JOOTRH facility was conducted. Systematic sampling was used to select the study participants while purposive sampling was used to select the nurses involved in key informant interviews. Semi-structured questionnaires and recording were used to collect data. Descriptive and logistic regression was conducted for the quantitative data. Qualitative data was transcribed, coded and analyzed thematically. Results The study found that 39% of the participants had the correct knowledge and practice of BSE. An increase in education level was significantly associated with correct knowledge and practice of BSE. Single participants (aOR = 0.6, 95%CI [0.36–0.99]), participants who heard about BSE from media (aOR = 0.51, 95%CI [0.27–0.97]) and friends (aOR = 0.25, 95%CI [0.09–0.69]) were less likely to report correct knowledge and practice of BSE. Conclusion There needs to be concerted effort to raise the knowledge and practice of BSE by the national and county governments, stakeholders and health practitioners for the early detection of breast cancer.