Accurate characterization of the aggregation and assembly of molecules at surfaces and interfaces is essential to understand molecular interactions and functionalities of molecular devices at interfaces. Sub-monolayer sensitive in situ methods are highly desirable to access the aggregation processes. In this study, using malachite green (MG, a commonly used conjugated molecule) chloride as a model, we demonstrate that analyzing the stability of second harmonic intensity is a powerful and sensitive method for determining the inhomogeneity of dye molecules at liquid interfaces in situ. The fluctuations correlate with surface domains and hence can be used to characterize the distributions of MG and MG aggregates. Moreover, we found that interfacial aggregation below the resolution of Brewster angle microscopy could still be detected with second harmonic generation analysis. Overall, this approach provides insight concerning the surface behavior of MG, and could be used for the aggregation analysis of other conjugated molecules.