Quartz-enhanced conductance spectroscopy is developed as an analytical tool to investigate dynamic nanomechanical behaviors of polymer wires, in order to determine the glass transition temperature (Tg). A polymethyl methacrylate (PMMA) microwire with a diameter of 10 μm was bridged across the prongs of a quartz tuning fork (QTF). With the advantage of QTF self-sensing as compared with micro-cantilevers or other resonators, the resonance frequency and Q factor can be directly determined by means of its electrical conductance spectra with respect to the frequency of the external excitation source (dI/dV vs f), and therefore, no optical beam is required. The Tg of the PMMA microwire was determined by the maximum loss modulus of the QTF, calculated from the resonance frequency and the Q factor as a function of temperature. The measured Tg of the PMMA is 103 °C with an error of ±2 °C. Both heating/cooling and physical aging experiments were carried out, demonstrating that the technique is both reversible and reproducible.