A novel plant tissue-based chemiluminescence (CL) biosensor for glycolic acid combined with flow injection analysis is proposed in this paper. The spinach tissue acts as the molecular recognition element. Glycolic acid is oxidized by oxygen under the catalysis of glycolate oxidase in the tissue column to produce hydrogen peroxide, which can react with luminol in the presence of peroxidase of spinach tissue to generate a CL signal. The CL emission intensity was linear with glycolic acid concentration in the range of 4