Understanding doping in polymer semiconductors has important implications for the development of organic electronic devices. This study reports a detailed investigation of the doping of the poly(3-hexylthiophene) (P3HT)/Nafion bilayer interfaces commonly used in organic biosensors. A combination of UV–visible spectroscopy, dynamic secondary ion mass spectrometry (d-SIMS), dynamic mechanical thermal analysis, and electrical device characterization reveals that the doping of P3HT increases with annealing temperature, and this increase is associated with thermally activated interdiffusion of the P3HT and Nafion. First-principles modeling of d-SIMS depth profiling data demonstrates that the diffusivity coefficient is a strong function of the molar concentration, resulting in a discrete intermixed region at the P3HT/Nafion interface that grows with increasing annealing temperature. Correlating the electrical conductance measurements with the diffusion model provides a detailed model for the temperature-modulated doping that occurs in P3HT/Nafion bilayers. Point-of-care testing has created a market for low-cost sensor technology, with printed organic electronic sensors well positioned to meet this demand, and this article constitutes a detailed study of the doping mechanism underlying such future platforms for the development of sensing technologies based on organic semiconductors.