To reduce anti-nutritional factors in plant protein sources for fish meal replacement in fish feeds, cottonseed and soybean meal (CS) were fermented with Aspergillus oryzae. A feeding trial was conducted to verify the effects of fermented CS (FCS) with phytase supplementation on gossypol detoxification, phosphorus digestibility, antioxidant activity, and growth performance of juvenile olive flounder over 10 weeks. Four diets were formulated to replace 0, 30, or 40% fish meal protein with CS or FCS (designated as CS0, CS30, FCS30P, and FCS40P). Phytase (1,000 FTU/kg) was added to FCS30P and FCS40P. The microbial fermentation significantly increased dietary total polyphenols and consequently led to higher DPPH radical-scavenging activities in fish feed and fish tissue. Dietary and liver gossypol concentrations were dramatically decreased by the fermentation process. Phosphorus digestibility was significantly increased in fish fed the FCS40P diet. However, growth performance decreased in fish fed FCS diets. This study demonstrates that the fermentation process and phytase supplementation can improve the phosphorus availability of plant protein sources in fish. The fermentation of CS by A. oryzae could increase antioxidant activities in feed and fish and effectively degrade toxic gossypol in cottonseed meal.