Clarifying the atomic configuration of step edges on a rutile TiO2 surface is crucial for understanding its fundamental reactivity, and the direct observation of atomic step edges is still a challenge. AFM is a powerful tool for investigating surface structures with true atomic resolution, and it provides the opportunity to resolve the real structure of step edges with improved techniques. In this work, we successfully imaged the atomic configuration of 001 and 1-11 step edges on the surface of rutile TiO2(110)-(1 × 1), and we present the direct observation of oxygen vacancies along the 1-11 step edges, indicating that one 1-11 step edge site corresponds to one oxygen vacancy using AFM. We also made use of the simultaneous AFM/STM measurements to explore the electronic structure of step edges, which enhanced the evidence of oxygen vacancies existing along the 1-11 step edges and further demonstrated that the 001 step edge was terminated by an O row. The effect of the reduced 1-11 step edges was explored by probing the O2 adsorption and the nucleation behavior of gold clusters. It was found that oxygen vacancies along the 1-11 step edges could contribute to O2 dissociative adsorption and there was no obvious difference compared with the oxygen vacancies on the flat terrace. The reduced step edge and terrace likewise acted as nucleation and growth sites for gold atoms/nanoparticles, in line with previous reports. The present study provides a complete characterization of the atomic configuration of the step edges on the TiO2(110) surface and plays an important role in investigating the surface chemistry of metal oxides.