To explore the tolerance mechanism of Medicago sativa under oil pollution, this study adopted the method of ultrasonic crushing and solubilization to prepare a mixed solution of three organic compounds (Dodecane, n-Hexadecane, and n-Tetracosane) at a concentration of 1% to simulate saturated alkanes and treat Medicago sativa seedlings. Samples were taken from plants exposed to pollution for 0h, 6h, and 24h for transcriptomic analysis. The results showed that a total of 1431 DEGs were obtained. GO enrichment analysis indicated that these DEGs were mainly involved in protein binding, metabolic pathways, and catalytic activity. KEGG enrichment analysis showed that DEGs were mainly enriched in plant pathogen interaction, MAPK signaling pathway and photosynthetic biological carbon fixation pathway. qRT-PCR was further used to verify the reliability of the results. This study provides theoretical basis for exploring the mechanism of plant degradation and tolerance of saturated petroleum hydrocarbon in crude oil pollution, as well as subsequent screening and cultivation of petroleum-tolerant plants through transcriptome analysis of Medicago sativa treated with saturated alkanes.