Flow injection analysis (FIA) is widely used in drug screening, neurotransmitter detection, and water analysis. In this study, we investigated the electrochemical sensing performance of glassy graphene electrodes derived from pyrolyzed positive photoresist films (PPFs) via rapid thermal annealing (RTA) on SiO/Si and polycrystalline diamond (PCD). Glassy graphene films fabricated at 800, 900, and 950 °C were characterized using Raman spectroscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM) to assess their structural and morphological properties. Electrochemical characterization in phosphate-buffered saline (PBS, pH 7.4) revealed that annealing temperature and substrate type influence the potential window and double-layer capacitance. The voltammetric response of glassy graphene electrodes was further evaluated using the surface-insensitive [Ru(NH)] redox marker, the surface-sensitive [Fe(CN)] redox couple, and adrenaline, demonstrating that electron transfer efficiency is governed by annealing temperature and substrate-induced microstructural changes. FIA with amperometric detection showed a linear electrochemical response to adrenaline in the 3-300 µM range, achieving a low detection limit of 1.05 µM and a high sensitivity of 1.02 µA cm/µM. These findings highlight the potential of glassy graphene as a cost-effective alternative for advanced electrochemical sensors, particularly in biomolecule detection and analytical applications.