Some water-insoluble substances found in the mother liquor of vitamin C recovery production test. Block the resin column influenced the recovery of mother liquor, and so their composition and content need to be determined by identification in the experiment. Firstly the water-insoluble substance was refined, then the quantitative and qualitative analysis was carried out by IR and HPLC. The results show that the insoluble substance contained 95% CaC2O4·H2O which was produced by oxalic acid whose content in the mother liquor is very small reacted with Ca2+. The successful identification of the water-insoluble substance could help improvement of the new recovery process.