Background: Enamel deproteinization using sodium hypochlorite entails the oxidation of organic components, resulting in the dissolution of proteinaceous components and subsequent cleansing of the enamel substrate. This oxidative treatment augments surface energy and promotes favorable interfacial interactions, thereby enhancing the adhesion and performance of dental materials, ultimately leading to better clinical outcomes. Aim: To evaluate the effectiveness of enamel deproteinization prior to etching on the retention of pit and fissure sealants (PFS) at different time intervals among 6–14-year-old children. Objective: The objective of this study was to evaluate the effect of 5.25% sodium hypochlorite on the clinical performance and retention of pit and fissure sealants in permanent molars. Materials and methods: A double-blind randomized controlled trial using a split-mouth design was conducted for a period of 1 year. The clinical trial registry was done CTRI/2023/06/053542. About 20 children aged 6–14 years, who required bilateral PFS for permanent molars, were included and divided into 20 units for each group. The contralateral side served as the control for the same patient. Randomization was performed using computer-generated numbers (Randomizer.com). Treatment was divided into two groups: group I: pit and fissure sealant with enamel deproteinization prior to etching, group II: pit and fissure sealant without enamel deproteinization. A blinded evaluator assessed the clinical outcome using modified Simonsen's criteria. Results: At the end of 12 months of follow-up, the results in the current study show that there is a statistical significance between groups I and II. In group I, only 10 teeth have experienced the event, while in group II, it is maximum, accounting for 33 teeth. It was found that the retention rate in group I is 88.1%, while in group II, it is 60.0%. Thus, we can conclude that the retention of pit and fissure sealant in group I is significantly better than in group II. Conclusion: The present trial yielded notable enhancements in the retention outcomes of PFS following the incorporation of an adjunctive procedure involving enamel deproteinization utilizing 5.25% sodium hypochlorite, combined with intermediate bonding, over a 12-month observation period. This approach holds promise as a strategic intervention for augmenting adhesion and optimizing the efficacy of PFS as a preventive measure.