This research aims at studying the temporal distribution of fish assemblage in the Karnafully river estuary, Bangladesh. To study the fish assemblage catch from Estuarine Set Bag Net (Behundijal) as well water samples were collected in four seasons (winter, pre-monsoon, monsoon and post-monsoon) from March 2013 to February 2014. Multivariate analyses were performed using the software PRIMER V₆ and Canonical Correspondence Analysis (CCA) was done using the PAST software. The present study identified 41 fish species, where Harpadon nehereus (20.54%) was the most dominant fish species. The Shannon-Wiener diversity index (2.67±0.05), Margalef richness index (5.14±0.64), Pielou’s evenness index (0.96±0.007) was high, while Simpson dominance index (0.95±0.002) was low in the monsoon season, which indicates monsoon had high fish diversity than the other seasons. At the similarity of 50.5%, three groups were attained while, monsoon-2 showed separate clustering from other groups. The Non-metric Multidimensional Scaling (nMDS) showed 20% similarity in all seasons based on Bray-Curtis similarity matrix. The CCA ordination indicated that salinity and DO were the two most important environmental parameters shaping the fish assemblage structure in the Karnafully river estuary.