To detect oxidative capacity and redox potential of organism simultaneously, a surface-enhanced Raman scattering-active microneedle was fabricated by integrating oxidative capacity and redox potential surface-enhanced Raman scattering probes in two grooves on an acupuncture needle, respectively. Different redox pairs solutions were used to assess the detection feasibility of the surface-enhanced Raman scattering-active microneedles. Oxidative capacity surface-enhanced Raman scattering probes, based on p-phenylenediamine, can discriminate minor changes in oxidative capacity, and redox potential surface-enhanced Raman scattering probes, based on an anthraquinone derivative, distinguish changes in redox potential of different redox pairs. Rats with or without high glucose-induced stress were utilized to evaluate the detection feasibility of the surface-enhanced Raman scattering-active microneedles in vivo. In different tissues, the two parameters show slight discrepancies. In the muscles of rats with high glucose-induced stress, no correlation was observed among oxidative capacity, redox potential, and blood glucose levels. The surface-enhanced Raman scattering-active microneedles would be a versatile tool for researches of oxidative capacity and redox potential in physiological and pathological processes.