Detection of methanol in commercial biodiesel is essential to evaluate the fuel quality associated with the matter of driving safety. Compared to the standard GC method, the fluorescent detection method for methanol is much simpler and faster, but still uncompetitive due to its low sensitivity. In this respect, we herein reported two flavonoid-based solvatofluorochromic probes by extending conjugation length to regulate the transition dipole moments, ensuring adequate detection sensitivities and discriminable fluorescent color change towards trace amounts of methanol in biodiesel. Spectroscopic studies revealed that the detection sensitivities of both probes were high and the detection limit for methanol in biodiesel could reach 0.041%, meeting the requirement of the commercial testing standard. The detection methods for methanol based on the above-mentioned probes were proven to be fast, stable and with acceptable veracity and reproducibility, especially suited for practical applications. This feasible design strategy by rationally regulating the transition dipole moments to tune the sensitivity of probes may inspire more related studies in the future.