Developing electrocatalysts with the trade-off between overall electrocatalytic performance and economic cost towards hydrogen evolution reaction (HER) is crucial for producing green hydrogen fuels. Although theoretical studies have reported the potential of two-dimensional (2D) MXenes for the HER, their electrocatalytic performance is still unsatisfactory for the particular application due to their low intrinsic active and MXene stacking. To address this problem, we herein modify the electronic structure and increase the interlayer spacing between 2D Ti3C2Tx MXenes through ammonia (NH3)-assisted hydrothermal approach. The effect of synthesis temperature on the nitrogen doping mechanism and electrochemical behaviors for the HER is systemically explored. As a result, the nitrogen (N)-doped Ti3C2Tx MXene fabricated at 160 °C exhibits the highest HER activity among all 2D N-doped Ti3C2Tx nanosheets with the overpotential/Tafel slope of 186.91 mVRHE/115.94 mVRHE dec−1 in an acidic solution. Due to the synergistic contribution of nitrogen, the N-doped Ti3C2Tx-160 °C has mass activity of 337.95 mA mg−1 at 0.3 VRHE, being 27.50-fold higher than that of pristine Ti3C2Tx. This work indicated that heteroatom doping is a facile but efficient approach for modulating the electronic structure and intrinsic activity of MXene-based materials for electrochemical reactions.