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Introduction: Thalassemia is the most common single gene disorder globally and has high burden in India. Education to eligible couples about the risk of conceiving a child with thalassemia is important for its prevention as well as for reducing psychosocial impact on them and improving family health. Objectives: To assess the knowledge, attitude, and practice regarding thalassemia among eligible couples residing in a slum of Kolkata and to find out the determinants of inadequate knowledge, negative attitude, and bad practice among them. Materials and Methods: A descriptive cross-sectional study was carried out in a slum in Kolkata, from October-December 2019 among 300 study population using a pre designed, pretested structured schedule by face-to-face interview method. All statistical analyses were conducted using SPSS version 16.0 and correlation of variables was seen using Pearson's Chi Square Test and Odds Ratio. Results: About 53.33% of the study population had adequate knowledge; 50.33% had positive attitude but only 9.67% had good practice. Conclusion: Eligible couples lack adequate knowledge on thalassemia; their attitude was positive but practice was bad. Significant determinants of inadequate knowledge were female gender, level of education and socio-economic status; of negative attitude-religion, level of education and socio-economic status whereas of bad practice – age, level of education and socio-economic status.