In this research, a new polymer substrate for glucose colorimetric sensing was developed with electrospun polycaprolactone (PCL) and 3,3’,5’,5’-tetramethylbenzidine (TMB) hybrid micro/nanofibers. Biodegradable polymer PCL and chromogen TMB were combined in electrospinning process so that fibers consisted with PCL and TMB with diameters ranging from several hundreds of nanometers to several micrometers were achieved. With the piling up of micro/nanofibers, a flexible substrate with porous inner structure was formed up. After air-plasma treatment and loading of enzymes for colorimetric reaction, the substrate could be used for colorimetric assay. Compared to conventional paper-based sensing substrates, the electrospun substrate achieved better immobilization of chromogen so that a higher sensitivity. Also, the morphology of electrospun substrate could be tuned to meet potential further sensing requirements.