To enrich high-quality mushroom cultivation substrates and expand the high-value utilization of reed straw, the raw material ratio for cultivation of Pleurotus ostreatus with reed base was studied by using test fungus ‘Xiahui No.1’. Seven modified substrates with 0%, 25%, 33%, 50%, 67%, 75%, and 100% reed substitution ratios were set up to cultivate the P. ostreatus, and the impact of the reed addition amount on the nutritional quality of P. ostreatus fruit bodies was analyzed. The zinc and selenium contents of P. ostreatus fruit bodies increased by more than 18.9% when reeds replaced 50% of wood chips compared to pure wood chips. In addition, their crude protein, medical amino acid, essential amino acid, and total amino acid contents increased by more than 10.5%, sweet and delicious amino acids increased by more than 11.6%. Similarly, selenium content increased by 65.9%, and zinc, iron, and vitamin B2 increased by more than 30.4% when reeds replaced 67% of wood chips compared to pure wood chips. In addition, medical amino acid, essential amino acid, and total amino acid contents increased by 3.3%, 14.9%, and 6.8%, respectively. Moreover, the score of ratio coefficient of amino acids, essential amino acid index, biological value, and nutrition index values were the highest among the seven modified substrates. The substrate with a 50% reed substitution ratio had higher medicinal value and better edible flavor. The amino acid composition of mushrooms grown with a 67% substitution ratio was closer to Food and Agriculture Organization (FAO) and World Health Organization (WHO) guidelines and egg model protein, with higher contents of multiple trace elements and better overall nutritional value. From the nutritional balance perspective, the reed substitution ratio in substrate should not be too high.