Plant pathogens continuously impair agricultural yields and food security. Therefore, the dynamic characterization of early pathogen progression is crucial for disease monitoring and presymptomatic diagnosis. Hyperspectral imaging (HSI) has great potential for tracking the dynamics of initial infected sites for presymptomatic diagnosis; however, no related studies have extracted fingerprint spectral signatures (FSSs) that can capture diseased lesions on leaves during the early infection stage in vivo or investigated the detection mechanism of HSI relating to the host biochemical responses. The FSSs denote unique and representative spectral signatures that characterize a specific plant disease. In this study, the FSSs of spot blotch on barley leaves inoculated with Bipolaris sorokiniana were discovered to characterize symptom development for presymptomatic diagnosis based on time-series HSI data analysis. The early spectral and biochemical responses of barley leaves to spot blotch progression were also investigated. The fullspectrum FSSs were physically interpretable and could capture the unique characteristics of chlorotic and necrotic tissues along with lesion progression, enabling the in situ visualization of the spatiotemporal dynamics of early plant–pathogen interactions at the pixel level. Presymptomatic diagnosis of spot blotch was achieved 24 h after inoculation—12 h earlier than the traditional polymerase chain reaction (PCR) assay or biochemical measurements. To uncover the mechanism of HSI presymptomatic diagnosis, quantitative relationships between the mean spectral responses of leaves and their biochemical indicators (chlorophylls, carotenoids, malondialdehyde (MDA), ascorbic acid (AsA), and reduced glutathione (GSH)) were developed, achieving determination coefficient of prediction set (R_p²) > 0.84 for regression models. The overall results demonstrated that, based on the association between HSI and in vivo planttrait alterations, the extracted FSSs successfully tracked the spatiotemporal dynamics of bipolaris spot blotch progression for presymptomatic diagnosis. Tests of this methodology on other plant diseases demonstrated its remarkable generalization potential for the early control of plant diseases.