pH sensors based on a nanostructured ion-sensitive field-effect transistor have characteristics such as fast response, high sensitivity and miniaturization, and they have been widely used in biomedicine, food detection and disease monitoring. However, their performance is affected by many factors, such as gate dielectric material, channel material and channel thickness. In order to obtain a pH sensor with high sensitivity and fast response, it is necessary to determine the appropriate equipment parameters, which have high processing cost and long production time. In this study, a nanostructured ion-sensitive field-effect transistor was developed based on the SILVACO technology computer-aided design (TCAD) simulator. Through experiments, we analyzed the effects of the gate dielectric material, channel material and channel thickness on the electrical characteristics of the nanostructured field-effect transistor. Based on simulation results, silicon nitride was selected as the gate dielectric layer, while indium oxide was chosen as the channel layer. The structure and parameters of the dual channel ion-sensitive field-effect transistor were determined and discussed in detail. Finally, according to the simulation results, a pH sensor based on the nanostructured ion-sensitive field-effect transistor was fabricated. The accuracy of simulation results was verified by measuring the output, transfer and pH characteristics of the device. The fabricated pH sensor had a subthreshold swing as low as 143.19 mV/dec and obtained an actual sensitivity of 88.125 mV/pH. In addition, we also tested the oxidation reaction of hydrogen peroxide catalyzed by horseradish peroxidase, and the sensitivity was up to 144.26 pA mol-1 L-1, verifying that the ion-sensitive field-effect transistor (ISFET) can be used to detect the pH of micro solution, and then combine the enzyme-linked assay to detect the concentration of protein, DNA, biochemical substances, biomarkers, etc.