It is of interest to in vivo monitor the co-dynamics of different substances. However, the tracking of multiple species is still challenging. In this work, we demonstrate an in vivo electrochemical method by using multi-potential step amperometry to in vivo detect ascorbic acid (AA) and oxygen (O2) simultaneously. In order to achieve good selectivity and high sensitivity for both AA and O2, we design a cobalt corrole [Co(tpfc)(py)2] (tpfc = 5,10,15-tris(penta-fluorophenyl) corrole, py = pyridine, denoted as Co-TPFC) and carbon nanotube nanocomposite to modify a carbon fiber microelectrode (Co-TPFC/MWNT/CFE). This Co-TPFC/MWNT/CFE exhibits excellent electrocatalytic properties towards the reduction of O2 preceding a 4e process and facilitates the oxidation of AA at low potential in the physiological environment. Based on this, we realize simultaneous detection of AA and O2 using two-potential steps (one cathodic (-0.2 V) and the other anodic (+0.05 V)) with 1 second step time. Both in vitro and in vivo experiments proved the feasibility of this method. This demonstrated strategy is useful for us to understand various physiological and pathological processes associated with O2 and AA co-dynamics, and also provides an idea for detecting multiple substances simultaneously.