Object To investigate the dependency of thermal expansion coefficient of DNA adsorption film on environmental conditions. Methods By treating DNA adsorption film as a macroscopic continuum film with prestrain, an equivalent composite beam model of DNA film-substrate was established to calculate the deflection of DNA-microcantilever beam under temperature loading; With Parsegian’s empirical potential describing the mesoscopic free energy of DNA adsorption film, the DNA liquid crystal-substrate multiscale deflection model, the thought experiment method and the equivalent deformation method are combined to establish the trans-scale relationship between the microstructure of DNA adsorption film and its macroscale mechanical properties. Thereout the thermal expansion coefficient of DNA adsorption film was predicted. Results Given the ionic strength, the thermal expansion coefficient of double-stranded DNA adsorption film ranges from 0.3×10?4 K?1 to 8.05×10?4 K?1, and that of single-stranded DNA adsorption film ranges from 1.28×10?4 K?1 to 9.33×10?4 K?1. Conclusions As a leading role in the competition of micro-interactions, the change of configurational entropy determines the dependency of thermal expansion coefficient of DNA adsorption film on environmental conditions; the thermal expansion coefficient of DNA adsorption film decreases with the increase of temperature or ion concentration or DNA packing density; in contrast with DNA molecules in bulk solution, the thermal expansion coefficient of DNA adsorption film on substrate is weakly correlated with temperature change, whereas strongly correlated with ion concentration change and packing density change, among of which the packing density change is the most sensitive factor. These results are useful for gene detection and its regulation, and provide reference for the evaluation of tissue organ performance in tissue engineering.