Recently reported nanofluidic diodes with highly nonlinear current-voltage characteristics offer a unique possibility to construct different biosensors. These sensors are based on local changes of the surface charge on walls of single conical nanopores induced by binding of an analyte. The analyte binding can be detected as a change of the ion-current rectification of single nanopores defined as a ratio of currents for voltages of one polarity, and currents for voltages of the opposite polarity. In this article, we provided both modeling and experimental studies of various biosensing routes based on monitoring changes of the rectification degree in nanofluidic diodes used as a biosensing platform. A prototype of a sensor for the capsular poly γ-D-glutamic acid (γDPGA) from Bacillus anthracis is presented. The nanopore used for the sensing was locally modified with the monoclonal antibody for γDPGA. The proof of principle of the rectification degree-based sensing was further shown by preparation of sensors for avidin and streptavidin. Our devices also allowed for determination of the isoelectric point of the minute amounts of proteins immobilized on the surface.