Objective To investigate the influence of the microscale attractive interaction on the elastic properties of DNA film in multivalent ion solutions. Methods Kornyshev's electrostatic zipper model was employed to describe the interaction energy between the DNA strands. The thought experiment method and macroscopic continuum bar model were combined to predict the stress-strain relationship, prestress, and elastic modulus of the DNA biofilm.Results Given the packing conditions, the DNA film exhibited a tensile prestress and negative elastic modulus. The prestress of the DNA biofilm ranged from -1.52 MPa to 1.17 MPa, and its elastic modulus ranged from -4.2 MPa to 64 MPa. Conclusions In contrast with monovalent solutions, the microscopic attractive interactions in multivalent solutions caused the elastic properties of the DNA film to exhibit a non-monotonous relationship with the variation in the packing density and salt concentration. The tensile elastic properties were significantly different from the compressive ones, and the tensile/compressive prestress as well as the positive/negative elastic modulus transformed each other. These results can contribute to understanding the mechanism of viral replication and provide references for gene detection and gene therapy.