Metalation reaction of metal-free phthalocyanine molecule with Co atom adsorbed on Au(111) surface has been studied in situ at single atom/molecule scale by scanning low-temperature tunneling microscopy (STM) experiment combined with simulations based on density function theory (DFT) calculations. Through manipulations using STM tip, we showed a controlled manner to have a single metal-free phthalocyanine molecule react with a Co atom into Co phthalocyanine molecule. In this reaction process, an intermediate state originating from π-d interaction between the metal-free phthalocyanine molecule and Co atom has been identified. Moreover, we also revealed that the redox reaction represented as bond breaking and bond forming relative to the Co and pyrrolic N atoms, not pyrrolic H atoms, is a key process for dehydrogenation and metalation reaction. Our DFT calculations provided theoretical supporting for the above conclusions, and further understanding for the related mechanisms.