Control of work function (WF) in graphene is crucial for graphene application in electrode material replacement and electrode surface protection in optoelectronic devices. Although efforts have been made to manipulate the effective WF of graphene to optimize its application, most studies have focused on graphene employed in static electrical contact interfaces. In this work, we investigated WF variations of supported single-layer graphene (SLG) in sliding electrical contact under ambient conditions, which was achieved by sliding an electrically biased conductive atomic force microscopy (cAFM) probe on the SLG surface. The effective WF, structural properties, and chemical compositions of rubbed SLG were subsequently measured by Kelvin probe force microscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy, respectively. We found that the effective WF of the rubbed SLG was governed by both the tunneling triboelectric effect (TTE) and tribochemical-induced surface functionalization. The TTE charges generated by the sliding cAFM probe tunneled through the structural defects of the SLG and were trapped underneath the SLG. The SLG will be either p-doped or n-doped depending on the type of TTE charges and the polarity of electric bias applied to the cAFM probe during the rubbing process. However, the applied electric bias also led to the electrolysis of a water meniscus formed at the cAFM probe-SLG contact, resulting in surface oxidation and the increase of SLG WF. Further absorption of ambient water molecules on the oxygenated functional groups gradually reduced the SLG WF. The influence of TTE and surface functionalization on the SLG WF depends on the magnitude and polarity of applied electric biases, relative humidity, and physical properties of the supporting substrates. Our results demonstrate that the effective WF of SLG in a sliding electrical contact interface will vary with time and might need to be considered for related applications.