This study aimed to investigate the impact of dietary selenium treatments on various sperm parameters and antioxidant responses in aged roosters to determine the relative bioavailability value (RBV) and optimize selenium supplementation. Over 40 days, starting from week 47 of age, the roosters were fed ten experimental diets, including a basal diet without selenium supplement and nine selenium-treated diets. These selenium-treated diets comprised three different selenium sources (selenium selenite: SS; SelPlex: Se-enriched yeast; selenium nanoparticles: SeNPs), each with three levels of selenium supplements (0.15, 0.30, and 0.45 mg/kg). Statistical analysis indicated significant treatment effects on all measured parameters except sperm volume. Sperm motility and viability increased linearly with higher dietary selenium levels. The relative bioavailability values (RBV) of SelPlex and SeNPs compared to SS were estimated using the slope ratio and exponential regression methods. Using the slope-ratio method, the RBV for sperm volume was 457% for SelPlex and 314% for SeNPs, compared to SS. Using exponential regression, the RBV of SelPlex and SeNPs for various parameters were as follows: for MDA (malondialdehyde), 260% and 317%; for TAC (total antioxidant capacity), 447% and 294%; for sperm morphology, 227% and 423%; and for sperm concentration, 346% and 298%, respectively. Principal component analysis revealed strong correlations between sperm motility, viability, and antioxidant parameters, with weaker associations observed between sperm volume and testosterone. Optimization using a desirability function identified 0.45 mg/kg selenium supplementation using SeNPs as optimal, maximizing sperm parameters and antioxidant responses while minimizing MDA and morphology. These findings offer valuable insights into effective selenium supplementation strategies to enhance avian reproductive health in aged roosters.