Objective To investigate elastic modulus of double-stranded DNA (dsDNA) biofilm adsorbed on microcantilever substrate. Methods Parsegian’s empirical potentials based on mesoscopic continuum liquid crystal theory was employed to describe the interaction energy among coarse-grained DNA cylinders; Monte Carlo method was used to simulate the distribution pattern of DNA chains before and after loading. The thought experiment method combined with the compression bar model in the sense of macroscopic continuum mechanics was adopted to predict the elastic modulus of DNA biofilm. Results The elastic modulus of dsDNA biofilm ranged from 0.1 MPa to 80 MPa. Conclusions It was found out that the classic hypothesis with uniform hexagonal pattern may underestimate the elastic modulus of DNA biofilm when compared with that in random pattern. Moreover, either the increase of packing density or the decrease of buffer salt concentration will help to enhance elastic modulus of DNA biofilm. These results have great significances in further understanding the mechanical properties and regulation rules of DNA biofilm related with clinical work.