TiO2 nanoparticle modified organ-like Ti3C2 MXene (TiO2-Ti3C2) nanocomposite has been synthesized and then used to immobilize hemoglobin (Hb) to fabricate a mediator-free biosensor. The morphology and structure of TiO2-Ti3C2 nanocomposite were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). Spectroscopic and electrochemical results revealed that TiO2-Ti3C2 nanocomposite is an excellent immobilization matrix with biocompatibility for redox protein, affording good protein bioactivity and stability. Due to the special organ-like hybrid structure of TiO2-Ti3C2, the direct electron transfer of Hb is facilitated and the prepared biosensors displayed good performance for the detection of H2O2 with a wide linear range of 0.1-380μM for H2O2 (sensitivity of 447.3μAmM-1cm-2), an extremely low detection limit of 14nM for H2O2. Especially, numerous TiO2 nanoparticles with excellent biocompatibility on the surface of the nanocomposite may provide a protective microenvironment for Hb to make the prepared biosensor improve long-term stability. The TiO2-Ti3C2 based biosensor retains 94.6% of the initial response to H2O2 after 60-day storage. TiO2-Ti3C2 nanocomposite could be a promising matrix for the fabrication of mediator-free biosensors, and might find wide potential applications in environmental analysis and biomedical detection.