The charge frustration with mixed-valence state inherent to LiTi2O4 that is found to be a unique spinel oxide superconductor, is the impetus for special attentions to unveil the clues of the pairing mechanism in unconventional superconductivity. Here we report a pronounced in-plane fourfold rotational symmetry of the superconductivity in high-quality single crystalline LiTi2O4 (001) thin films. Both the magnetoresistance and upper critical field under in-plane magnetic field manifest striking fourfold oscillations deep inside the superconducting state, and the anisotropy vanishes in the normal state, demonstrating that is intrinsic property of the superconducting phase. We attribute this behavior to the unconventional d-wave-like superconducting Cooper pairs in LiTi2O4, which is further supported by the scanning tunneling spectroscopy. Our findings demonstrate the unconventional character of the pairing interaction in three-dimensional spinel oxide superconductor and shed new light into understanding the pairing mechanism of unconventional superconductors.