Electrochemical biosensors are essential health monitors that aid in the detection and diagnosis of diseases. In this research, anatase titanium dioxide (TiO2) nanorods (TNR-A) were synthesized on a titanium (Ti) substrate in three stages, namely, hydrothermal, alkali, and heat treatments, and utilized as a modified electrode (TNR-A/Ti). The pretreatment of electrochemical reduction was adopted to increase the Ti3+ amount in TiO2 nanorod surfaces of the electrodes, thus improving electron transfer. The electrodes were characterized by X-ray diffractometry (XRD), fieldemission scanning electron microscopy (FESEM), and X-ray photoelectron spectroscopy (XPS). The electrodes after pretreatment (Ti3+-TNR-A/Ti) showed a better electrochemical response to hydrogen peroxide (H2O2), indicating that pretreatment improves the performance of electrodes. The assembled biosensor electrode (Nafion/HRP/Ti3+-TNR-A/Ti) exhibited a sensitivity of 6096.4 μA∙mM−1∙cm−2, a detection limit of 0.008 μM, a linearity of 0.04–700 μM, and an apparent Michaelis–Menten constant Kapp M of 0.0027 μM, which are higher than those in previous research studies on TiO2 or similarly nanostructured modified biosensor electrodes. This research could provide a potential competent method that can be used to modify electrodes for high-performance amperometric biosensors.