Biodiesel is a fuel composed of monoalkyl esters of long-chain fatty acids derived from renewable biomass sources. In this study, biomass waste pecan nutshell (PS) was attempted to be converted into microbial oil. For effective utilization of PS, sequential pretreatment with ethylene glycol-H2SO4-water (78:2:20, wt:wt:wt) at 130 A degrees C for 30 min and aqueous ammonia (25 wt%) at 50 A degrees C for 24 h was used to enhance its enzymatic saccharification. Significant linear correlation was obtained about delignification-saccharification (R (2) = 0.9507). SEM and FTIR results indicated that combination pretreatment could effectively remove lignin and xylan in PS for promoting its enzymatic saccharification. After 72 h, the reducing sugars from the hydrolysis of 50 g/L pretreated PS by combination pretreatment could be obtained at 73.6% yield. Using the recovered PS hydrolysates containing 20 g/L glucose as carbon source, microbial lipids produced from the PS hydrolysates by Rhodococcus opacus ACCC41043. Four fatty acids including palmitic acid (C16:0; 23.1%), palmitoleic acid (C16:1; 22.4%), stearic acid (C18:0; 15.3%), and oleic acid (C18:1; 23.9%) were distributed in total fatty acids. In conclusion, this strategy has potential application in the future.