Purpose: This study aims to evaluate the knowledge, attitude, and practices (KAP) regarding vision and eye screening of preschool children among staff at primary health centers (PHCs) in India. Methods: In 2024, a survey was conducted among PHC staff using a questionnaire with sections on knowledge (10 questions), attitude (5 questions), and practices (5 questions) related to preschool vision and eye screening. Responses were measured on a five-point Likert scale. The questionnaire's reliability was confirmed with a Cronbach’s alpha of 0.798. KAP scores were analyzed in relation to participants' demographic variables. Additionally, current and preferred sources of information were identified. Results: The survey included 101 health staff members (66 doctors and 35 nurses). The median (interquartile range) scores for knowledge, attitude, and practice were 4.1 (3.8; 4.3), 4.2 (4.0; 4.6), and 3.6 (3.0; 4.0), respectively. Doctors demonstrated significantly better knowledge (Mann-Whitney U test, P = 0.016) and attitude (Mann-Whitney U test, P = 0.019) compared to nurses. Staff over 40 years of age showed higher knowledge (Kruskal-Wallis H test, P = 0.035), attitude (Kruskal-Wallis H test, P = 0.017), and practice (Kruskal-Wallis H test, P < 0.001) scores. The primary source of information was medical education (51%), followed by eye care professionals (11.9%), online resources (12.9%), and social media (14.9%). Preferred information sources were medical journals (25.7%), eyecare training (22.8%), and eye professionals (33.7%). Conclusion: PHC staff in India generally have high knowledge and positive attitudes towards preschool vision and eye screening, though their practices are less consistent. Enhancing information dissemination through preferred channels could improve eye care practices for preschool children.