Nowadays, nanobiosensors are being intensively developed due to the potential possibilities of their use for early diagnosis of diseases. This interest is enhanced by the fact that, as is known, a pathological process at an early stage is characterized by the appearance of marker proteins at very low (10−15 M and lower) concentrations in blood. Highly-sensitive nanobiosensor systems (including those based on an atomic force microscope, AFM) allows one to detect proteins at such low concentrations. The influence of the charge generated in the analyte solution flowing through the biosensor injector into the measuring cell during measurements is considered to be an important factor conditioning such a high detection sensitivity. In the present study, it was demonstrated that the presence of an AFM chip (made of mica and graphite) near the nozzle of the injector supplying an analyte solution into the measuring cell of the AFM-based fishing system (AFM-based nanobiosensors) causes an increase in charge generation upon the injection of the solution. Moreover, the influence of polymer materials (which are widely used in nanobiosensors) and communications on charge generation in the flow-based section of AFM-based nanobiosensors was studied. A stimulating influence of a low (femtomolar) concentration of proteins on the charge generation in polymeric injectors of flow-based nanobiosensors was demonstrated. Besides, a stimulating influence of an external low-frequency AC electric field on the charge generation in the nanobiosensor injector was found. Measurements were carried out in the temperature range corresponding to the physiological temperature (35 °C).