Taking waste of nejayote as test material,the effect of liquid medium and culture conditions on the preparation of Pleurotus nebrodensia liquid spawn were studied by orthogonal and the single-factor test,liquid spawn application was compared.The results showed that Pleurotus nebrodensis liquid spawn was significantly better than the solid strains，the best medium for Pleurotus nebrodensia liquid spawn using waste of nejayote as raw material was glucose 3%，soybean meal 0.6%，vitamin B1 5 mg/L，MgSO4?7H2O 0.1%，KH2PO4 0.1%,nejayote making up 100%；the best Pleurotus nebrodensia fermentation conditions were initial pH 6.5，inoculum 10%，shaking speed 180 r/min.