Based on seasonal bottom trawl surveys in November 2015 (autumn), February (winter), May (spring) and August (summer) in 2016, the index of relative importance (IRI), Shannon index and Pianka index were used to analyze the breadth and overlap of spatio-temporal niche of major fish species in the Pishan waters off Zhejiang Province. Redundancy analysis and interspecific competition coefficient were used to examine the competition relationship and niche differentiation of those major fish species. The results showed that a total of 61 fish species were recorded throughout the year, which belonged to 13 orders, 29 families and 48 genera. A total of 19 species with IRI>100 were identified as major fish species. The temporal niche overlap value between Chelidonichthys kumu and Atule kalla was largest, indicating high temporal synchronization. Both the spatial niche overlap value and the spatio-temporal niche overlap value between Trpauchen vagina and Cynoglossus interruptus were largest, indicating that their spatial homology was high and that the use of spatial and temporal two-dimensional resource of both species were consistent. The percentage of species pairs with spatio-temporal niche overlap at the significant level (Qik>0.6) was only 5.8% in Pishan waters, indicating that the spatial and temporal distribution of those species were quite different. The overlap of spatio-temporal niche significantly varied across different seasons. The results of the interspecific competition coefficient were basically consistent with the niche overlap. Results of the redundancy analysis revealed the relationships between major fish species and environmental factors (temperature, salinity, dissolved oxygen), and further explained the niche diffe-rentiation among species.