This comprehensive study investigates the stage-wise infection dynamics of Nucleopolyhedrovirus (NPV) in fall armyworm, Spodoptera frugiperda (J. E. Smith) larvae across distinct stages of maize crop in Panchmahal and Mahisagar districts of Gujarat, India during the kharif season in 2021. The examination of month-wise mean data reveals a pronounced pattern of NPV prevalence, with the highest infection observed during the cob formation stage (28.74 %), followed by flowering and tasseling (20.23 %), and the vegetative stage (13.93 %). Block specific analyses highlight variations in infection rates, with notable differences between districts and stages. Correlation coefficient analysis indicates a significant and positive relationship between specific crop stages and NPV infection. Regression equations are established, demonstrating a positive increase in natural NPV infection during different crop stages. Weather parameter analysis reveals significant correlations between temperature, humidity, sunshine hours, and NPV infection. The phylogenetic analysis of NPV polyhedrin sequences indicates species-specific clustering within S. frugiperda and a broader genus-specific association. Motif pattern analysis identifies conserved motifs in NPV polyhedrins. Protein structure predictions and Molecular docking studies predicted interactions between polyhedrin and chitinase, suggesting a potential role in the insect's defense against NPV. The bioassay results indicated increased susceptibility of early larvae to NPV, emphasizing the potential for optimizing biocontrol strategies. Overall, this study contributes valuable insights into the ecological and molecular aspects of NPV infection dynamics, offering a significance understanding for developing effective pest management strategies and also underscores its significance in promoting sustainable agriculture.