Amperometric hydrogen peroxide (H2O2) and glucose biosensors based on unzipped carbon nanotubes with modified glassy carbon electrode (GCE) have been successfully fabricated via a facile electrochemical oxidative method. In this work, we investigated the feasibility of this new form of carbon nanomaterial as a substrate electrode material for fabricating sensitive platform for H2O2 and glucose sensors. For this purpose, the manganese oxide (MnO2)/unzipped single-walled carbon nanotubes (SWCNTs) film was synthesized by the cyclic voltammetry method. The developed sensing film, MnO2/unzipped SWCNTs/GCE, displayed a satisfactory analytical performance for H2O2, including a wide linear range of 2.0 × 10−6 to 5.0 × 10−3 M with a detection limit of 0.31 × 10−6 M (10.7 ppb). This film was further applied for glucose sensing with a linearity range of 0.01 to 1.2 mM with a correlation coefficient of 0.9822 in the physiological pH (7.4). This facile, fast, environmentally-friendly, and economical preparation strategy of carbon nanomaterial-based electrode materials opens up the possibility of developing high quality biocompatible hydrogen peroxide and glucose sensors.