On-site quantitative analysis of 2,4-dichlorophenoxyacetic acid (2,4-D) is of significant importance for addressing increasing concerns about public health and environmental quality. Here, a novel metal–organic framework (MOF) of Co-hemin is synthesized and first used for on-site colorimetric monitoring of 2,4-D. 2,4-D as an inhibitor of alkaline phosphatase could specifically suppress the production of ascorbic acid, which restrained in situ etching of Co-hemin and further triggered the colorimetric response. In the colorimetric assay, Co-hemin displayed good oxidase-like activity without addition of H₂O₂, which could avoid the shortcomings of H₂O₂ such as toxicity and instability. The Co-hemin biosensor exhibited a relatively low detection limit of 33 ng mL⁻¹ for 2,4-D by the UV method. Moreover, a smartphone based RGB analysis system for the sensitive detection of 2,4-D was developed, and exhibited a good linear relationship between the RGB model parameter and the concentration of 2,4-D. The operability and accuracy of the Co-hemin biosensor were confirmed by the quantitative determination of 2,4-D in real samples, such as serum and tap water. Also, the Co-hemin based colorimetric biosensor showed good selectivity and specificity. Moreover, the developed assays displayed good application in constructing complex logic gates. This work not only provided a portable and visual platform for on-site monitoring of 2,4-D, but also expanded application prospects in the field of complex biological analysis.