The growing demand for glycolate, fueled by economic development, requires the advancement of production methods. Escherichia coli (E. coli), a preferred host for glycolate production, has undergone extensive metabolic engineering to improve yield. Developing rapid and precise methods for quantifying glycolate concentration is essential for screening high-yielding strains. Here, we present the engineering of a novel circularly permuted green fluorescent protein (cpGFP)-based glycolate sensor, termed GLYCO. GLYCO exhibits high specificity (minimal interference from other metabolites), stability (no decrease in performance after 15 days at -80 °C), and ease of detection via fluorescence measurement, enabling effective in vitro glycolate quantification. GLYCO spans a quantification range from 10 μM to 1 mM, facilitating effective monitoring of glycolate production in metabolically engineered E. coli strains. This biosensor represents a significant advancement in the metabolic engineering toolkit, facilitating efficient detection and optimization of glycolate production in E. coli, with potential applications in industrial biotechnology.