Luminol and its derivatives are extensively used as chemiluminogenic substrates in bioimaging and biochemical analysis. Luminol reagents can typically emit blue chemiluminescence (CL), whose wavelength is normally outside the most sensitive detection range of human naked eyes and most CL analyzers with silicon-based charge-coupled device (CCD) detectors. Development of luminol analogues with longer wavelength emission is thus attractive. Herein, four new phthalhydrazide CL probes (GL-1/2/3/4) have been prepared through the derivatization of luminol. The most promising one, 5-(4-hydroxy-1,3-dioxoisoindolin-2-yl)-2,3-dihydrophthalazine-1,4-dione (GL-1), emits bright green CL upon oxidation and shows enhanced CL performance compared to its parent luminol. Bloodstain imaging, horseradish peroxidase (HRP)-based immunoassay, and the analysis of glucose/glucose oxidase reaction have been performed using the GL-1 reagent. These results indicate that GL-1 is a new chemiluminogenic luminol analogue with great potential in real analytical applications and will be an alternative to replace luminol in practical CL analysis.