Litters of 3 types of grasses including Medicago sativa, Artemisia gmelinii, and Coronilla varia were mixed with 45.37 g/kg petroleum-contaminated soil with or without urea fertilization (the C:N of soil was adjusted to approximately 25:1) in the study. We aim to investigate the effects of plant litters and nitrogen fertilization combination on remediation of petroleum-contaminated soils. The mixtures were incubated at 20-25℃ with consistent soil moisture for 180 days. The microbial populations, the activities of 11 types of hydrolases and oxidoreductases, and contents of available N, P and K in soils were determined to analyze the individual or the combined effects of plant litter and urea on the bio-chemical properties of petroleum-contaminated soils. The results indicated that (1) all types of grass litters tested here significantly increased the populations of actinomycetes and fungi, the activities of sucrase, amylase, carboxymethyl cellulase, xylanase, urease, protease, alkaline phosphatase, polyphenol oxidase, catalase and peroxidase, and the contents of available N, P and K in petroleum-contaminated soils, while they significantly accelerated the degradation of total petroleum hydrocarbons. (2) Generally, urea application facilitated the remediation of grass litters on petroleum-contaminated soils. However, it might impair the positive effects of litter treatments on the activities of xylanase, urease, protease and dehydrogenase, and the microbial populations in petroleum-contaminated soils. (3) Sole litter treatment could remarkably improve the biochemical properties of petroleum-contaminated soils, especially with high N and P contents, lower C/N, C/P ratios, low lignin and polyphenols contents litters. In the case that the remediation was exigent, appropriate urea fertilization could be a feasible approach to accelerate the remediation effects of grass litters on petroleum-contaminated soils, whereas taking its negative effects on above-mentioned biochemical soil properties in account.