Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons and the accumulation of alpha-synuclein. Glutathione (GSH), a key antioxidant, is significantly depleted in PD patients. This study presents a dual-mode detection strategy for selectively determining GSH using a single probe. A series of "turn-on" electrochemical and fluorescent probes were developed, with resorufin (Re) serving as the reporting unit and featuring specific GSH recognition sites. Among these, the 7-(3,5-dinitrophenoxy)-3H-phenoxazin-3-one (Re-DNP) probe was selected for its high selectivity as both a fluorescent and electrochemical probe. Its response to GSH was superior in comparison to that observed for hydrogen sulfide (H2S) and cysteine (Cys). For electrochemical detection using screen-printed carbon electrode (SPCE)/carbon nanotube (CNT) modified electrodes, the detection limit for GSH was 5 μM, with a linear range of 25-500 μM. In fluorescence detection, the probe produced a 78-fold increase in emission at 630 nm in the presence of GSH, with a strong linear correlation between fluorescence intensity and GSH concentration in the range of 10-700 μM, and a detection limit of 2 μM. When applied to real clinical serum samples, the probe demonstrated significantly lower GSH levels in both PD mice and human patients compared to healthy controls. This dual-mode detection method provides a sensitive and accurate tool for GSH detection, with potential applications in understanding GSH's role in PD and related neurodegenerative diseases.