Intermittent preventive therapy during pregnancy with Sulphadoxine Pyrimethamine (IPTp-SP) is one of the potent strategies for preventing malaria in pregnancy (MiP). Factors such as the pregnant woman’s knowledge and very importantly perception or belief about IPTp-SP remains key determinant of IPTp-SP uptake. This study assessed the knowledge and perception of nursing mothers and their uptake of IPTp-SP during pregnancy in the Ho Teaching Hospital. We administered a close-ended questionnaire to 303 nursing mothers and obtained their sociodemographic details as well as information on their knowledge and perception of IPTp-SP utilization. We also reviewed the nursing mothers’ antenatal care (ANC) booklets to ascertain the number of times IPTp-SP was taken during pregnancy. Pearson chi-square was used to determine the association between the sociodemographic variable and the categories of knowledge, perception, and uptake of IPTp-SP. Analysis, was done using SPSS, and the p-value of less than 5% was considered statistically significant. Of the 303 nursing mothers sampled in this study, 265(87.5%) had heard about IPTp-SP of which 138(52.1%) had average knowledge of IPTp-SP. A total of 168(63.4%) had poor perception, and 168(64.6%) had adequate uptake (3–4 doses) of IPTp. Education was significantly associated with perception and uptake, with the majority of women who demonstrated excellent perception were those who had tertiary education (7, 6.35%, p = 0.05), and the majority who demonstrated excellent uptake (5 completed doses) were women who had tertiary education (47, 37.9%, p = 0.01). While knowledge was average, perception of IPTp-SP was poor for the majority of the nursing mothers which might have hampered their uptake of IPTp-SP during pregnancy. It is important that continuous assessment of the individual factors affecting the uptake of IPTp-SP be done regularly to curb the negative influences on the uptake of IPTp-SP.