The inhibitory potential of allelopathic plants is the subject of increasing research attention for their application in weed management. The sugarcane leaf is an agricultural waste product that has been reported to have allelopathic potential. Therefore, the present study determined the optimal organic solvent system and fractionation procedure to enhance the quantity of this extract and its allelopathic efficiency. Sugarcane leaves were extracted using five ethanol/water solvent ratios (v/v): 00:100, 25:75, 50:50, 75:25, and 100:00. Their allelopathic effects on seed germination and seedling growth were assayed in two major weeds, Echinochloa crus-galli (L.) Beauv. and Amaranthus viridis L. The results showed that the extract concentration, solvent ratio, and their interaction significantly inhibited the growth parameters in A. viridis. Consequently, a crude ethanol/water ratio of 00:100 was used to separate the active fraction via acid-base solvent partitioning. The acidic fraction (AE) exerted the greatest inhibitory effect and completely (100%) inhibited A. viridis at all concentrations, followed by the original crude fraction, neutral fraction, and aqueous fraction. Moreover, all of the fractions had selective effects, inhibiting A. viridis much more than E. crus-galli in the laboratory tests. The chemical analysis using gas chromatography/mass spectrometry indicated that the AE fraction contained 20 different compounds. The five major compounds included alkaloids, organic acids, and phenols. Therefore, the AE fraction was selected for formulation in a concentrated suspension and tested for its herbicidal characteristics. The formulation exhibited early post-emergence activities and had a stronger effect on A. viridis compared to E. crus-galli. The physiological mechanism of the formulation was tested against A. viridis. The thiobarbituric acid reactive substances and H2O2 occurred in the A. viridis leaf, which suggests lipid peroxidation and cell disruption.