The technique of self-assembled monolayers (SAMs) is frequently applied for grafting functional groups or area-selective deposition of thin films on a material surface. The formation and quality of SAMs are fundamentally determined by thermodynamic data, which are difficult to measure with available experimental methods. This work quantitatively extracted thermodynamic parameters including ΔH°, ΔG°, and ΔS° during the SAMs construction process with an ultrasensitive resonant microcantilever as molecule-surface interactions real-time recording tool. By correlating the thermodynamic parameters with self-assembling temperatures, a new thermodynamic phase-like transition effect of molecular self-assembly has been first revealed. The sharp transition of the thermodynamic parameters defines the critical condition for SAMs formation. The thermodynamic data further provide optimized reaction conditions for constructing high-quality SAMs. The explored quantitative thermodynamic analysis method not only plays as criterion for SAM growth but also helps to fundamentally elucidate physicochemical mechanism of spontaneous self-assembly.