Electrochemical biosensors are essential health monitors that aid in the detection and diagnosis of diseases. In this research, anatase titanium dioxide (TiO 2 ) 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 Ti 3+ amount in TiO 2 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 (Ti 3+ -TNR-A/Ti) showed a better electrochemical response to hydrogen peroxide (H 2 O 2 ), indicating that pretreatment improves the performance of electrodes. The assembled biosensor electrode (Nafion/HRP/Ti 3+ -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 K app M of 0.0027 µM, which are higher than those in previous research studies on TiO 2 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.