In this paper, a dual-functional probe, 2-(benzothiazol)-4-(3-hydroxy-4-methylphenyl) imino phenol (BHMH), was synthesized and characterized for the simultaneous detection of Cu2+ and Fe3+ in dimethyl sulfoxide/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (DMSO/HEPES) (1:4, v/v, pH = 6.0). The limits of detections (LODs) for Cu2+ and Fe3+ were 9.05 and 48 nM, respectively. Based on the competitive coordination, the complex BHMH-Cu2+/Fe3+ exhibited good sensitivity and selectivity for glyphosate. The LODs of BHMH-Cu2+ and BHMH-Fe3+ for glyphosate were 0.41 and 0.63 μM, respectively. The probe quantitatively detected glyphosate in tap water, Songhua River water, local water and soil, and food samples. The colorimetric on-site glyphosate sensing through the probe BHMH-Cu2+ was also studied based on smartphones. BHMH and BHMH-Cu2+/Fe3+ exhibited outstanding imaging capabilities for Cu2+, Fe3+, and glyphosate in living cells with low cytotoxicity, especially the first time for glyphosate.