We examined the glass transition behaviors of a series of homopolymers and random copolymers, poly(methyl methacrylate) (PMMA), poly(ethyl methacrylate) (PEMA), and poly(methyl methacrylate-random-ethyl methacrylate) (P(MMA-r-EMA)), in films deposited by two conventionally employed methods, spin-coating and the Langmuir-Blodgett (LB) method, exhibiting uncontrolled and controlled chain conformations at the molecular level, using spectroscopic ellipsometry measurements. The processing parameters of the LB method (surface pressure, deposition rate, and number of deposited layers) were controlled and the films were compared with those made by spin-coating to gain a deeper understanding of how the glass transition temperature is affected by the processing conditions. Our results strongly suggest that the glass transition temperature of polymer films follows typical thermal behaviors of corresponding bulk samples, even though they are deposited using the LB method. The bulk glass transition temperature (T-g) was controlled to further investigate how the deposition temperature affects the thermal properties of polymer films by adjusting the composition of P(MMA-r-EMA) random copolymers. The molecular layer by layer deposition using the LB method at temperatures ranging from 0.76 T-g to 0.87 T-g, where the small molecule glass films by physical vapor deposition have extraordinary high density, demonstrated glass transition behavior identical to that of the bulk samples. The thermal stability of the polymer films deposited using the LB method could not be compared to that of the small molecule films with similar deposition temperatures relative to T-g, as the inevitable trapping of water molecules during LB deposition resulted in low density and high apparent fictive temperature of LB deposited films.