Background <p>Enamel pretreatment with a deproteinization agent enhances fissure sealant bonding. This in vitro study investigates the effect of surface preparation methods and thermal cycling on the shear bond strength of fissure sealants applied to permanent teeth.</p> Methods <p>Sixty sound human third molars were randomly divided into ten groups (<i>n</i> = 6). Groups 1–5 received different surface treatments (control, HOCl 15&#xa0;s, HOCl 30&#xa0;s, NaOCl 60&#xa0;s, and bromelain (plant-based agent) 60&#xa0;s), while Groups 6–10 were subjected to identical protocols with the addition of 10,000 thermal cycles. All samples underwent shear bond strength testing. Statistical analysis was performed using IBM SPSS Statistics version 23.0 (IBM Corp., Armonk, NY, USA), with <i>p</i> &lt; 0.05 considered statistically significant.</p> Results <p>Surface preparation methods had no significant effect on shear bond strength (<i>p</i> = 0.431), while thermal cycling significantly reduced bond strength across all groups (<i>p</i> = 0.022). No interaction was found between the two factors (<i>p</i> = 0.480). A decreasing trend was observed within thermocycled groups: sodium hypochlorite &gt; bromelain &gt; hypochlorous acid (30&#xa0;s) &gt; hypochlorous acid (15&#xa0;s) &gt; control.</p> Conclusions <p>Within the limitations of this in vitro study, surface deproteinization did not statistically significantly increase shear bond strength compared to the control group. However, deproteinization agents showed a defining trend toward better preservation of bond strength stability following thermal cycling. Given their biocompatibility and favorable performance, bromelain and hypochlorous acid can be considered promising alternatives to sodium hypochlorite, but further clinical studies are needed to optimize application protocols and validate their long-term efficacy in pediatric dentistry.</p>