A spent Auricularia auricular substrate was oxidatively degraded using aqueous hydrogen peroxide/acetic anhydride to obtain a soluble portion (SP). The SP was sequentially eluted by using a polyamide-packed column with petroleum ether (PE), chlorine dioxide solution (CDS), ethyl acetate (EA), and mixed solutions of PE with CDS, and CDS with EA at different volume ratios, yielding 11 eluate fractions (F1–F11). In gas chromatography/mass spectrometry (GC/MS), 19 elution fractions (EF1–EF19) with similar chemical compositions were obtained. 136 compounds were identified in SP and EF1–19. Among these, seven compounds were completely separated: (r-(R*, R*))-2-acetoxy-3-hydroxysuccinic acid (AHSA), ribitol, tartaric acid (TA), 2-methyl-2-propenate-1,2-ethanediylester (MPEE), succinic acid (SA), acetoxyacetic acid (AAA), and dihydro-2,5-furandione (DHFDO). The purities of AHSA, ribitol, TA, MPEE, and DHFDO were 100.0, 100.0, 100.0, 96.0, and 99.0%, respectively, whereas those of SA and AAA were 100% after recrystallization. A series of carboxylic acids was enriched from SP and identified using a GC/MS system.