Ion-Sensitive Field-Effect Transistors (ISFETs), which were initially proposed a little more than 50 years ago, are currently among the most widely used electrochemical biosensors. In this work we propose a numerical simulation methodology for nanoscale ISFETs that is based on band-to-band tunneling conduction mechanism. The proposed method is based on the combination of the analytical equations of Tunneling Field-Effect Transistors (TFETs) with the Gouy–Chapman–Stern model equations of ISFETs. We then get a system of equations of Ion-Sensitive Tunneling Field-Effect Transistors (ISTFETs) that can be solved iteratively to produce output current and sensor sensitivity. The simulation is implemented using MATLAB software tool. The simulation results are verified by comparing the results of the developed code with Silvaco TCAD simulation software. The simulation code is then used for the optimization of the sensitivity and linearity of nanoscale ISFETs. We investigate the effect of various parameters, such as drain current level, reference voltage, gate-insulator thickness, substrate thickness, and temperature, on the sensor performance. Additionally, the effect of using different gate-insulator materials is inspected by comparing three different insulator types: SiO2, Al2O3, and HfO2. The simulation method can be applied to both single-gate and double-gate devices and serves as a guide for the design and optimization of nanoscale ISFETs.