Background: Coix (Coix lacryma-jobi L.) is cultivated as medicine and food homologous resources containing various active components. As one of its main ingredients, coixol possesses the biological activities of fever reduction, pain relief, tumor suppression, blood glucose, and pressure decrease. However, the biosynthesis mechanism of coixol in coix is still unclear. Methods: In this study, different dosages of salicylic acid (SA) were applied to coix plants, and the contents of coixol from different parts were detected by an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). The biosynthesis pathway of coixol was determined by high-throughput transcriptome sequencing analysis and the genes were then verified by qRT-PCR. Results: SA treatment significantly increased plant height, root length, and fresh weight and increased the coixol contents in the root, stem, leaf, and seed. In total, eight enzyme-encoding genes were screened out as the key genes in the biosynthesis of coixol. The bioaccumulation of coixol was mainly through benzoxazinoid biosynthetic metabolic pathway (ko00402). Conclusions: These findings not only pointed the way for increasing the content of coixol in cultivation but also provided a reference for further elucidation of the gene functions involved in the bioaccumulation of coixol.