Efficient detection of gonadotropin-releasing hormone analogues (GnRH-a) in fish is essential for monitoring pharmacokinetics and improving hormone-based breeding protocols in aquaculture. This study reports the development of a peptide-based indirect ELISA (iELISA) for detecting salmon GnRH-a (sGnRH-a) in fish sera and investigates the role of chitosan, a biological macromolecule, in enhancing hormone retention. Polyclonal antibodies were successfully raised in rabbits using a BSA-conjugated sGnRH-a peptide. The iELISA was optimized and validated, exhibiting 97.9 % sensitivity, 95.8 % specificity, and an AUC of 0.982. Cross-reactivity analysis revealed only 5.6 % reactivity with the related peptide, chicken GnRH-a. The assay was applied to Cyprinus carpio and Labeo catla, revealing that chitosan nanoconjugation extended sGnRH-a detectability in serum from 30 min to 1 h post-injection. Molecular docking using AutoDock Vina revealed a stable chitosan-sGnRH-a complex with a binding energy of -8.4 kcal/mol, supported by hydrogen bonding and hydrophobic interactions. These findings provide mechanistic insight into the sustained-release behaviour of chitosan nanocarriers. This is the first ELISA reported for sGnRH-a detection in fish sera, offering a sensitive, non-radioactive, and practical tool for hormone monitoring. The study highlights the utility of chitosan as a macromolecular delivery vehicle, contributing to improved reproductive management strategies in aquaculture.