Many DNA-based devices need to build stable and controllable DNA films on surfaces. However, the most commonly used method of film characterization, namely, the probe-like microscopes which may destroy the sample and substrate. Surface Forces Apparatus (SFA) technique, specializing in surface interaction studies, is introduced to investigate the effects of DNA concentration on the formation of single-stranded DNA (ss-DNA) film. The result demonstrates that 50 ng/μL is the lowest concentration that ss-DNA construct a dense layer on mica. Besides, it is also indicated that at different DNA concentrations, ss-DNA exhibit diverse morphology: lying flat on surface at 50 ng/μL while forming bilayer or cross-link at 100 ng/μL, and these ss-DNA structures are stable enough due to the repeatability even under the load of 15 mN/m. At the same time, an obvious adhesion force is measured: −6.5 mN/m at 50 ng/ μL and −5.3 mN/m at 100 ng/μL, respectively, which is attributed to the ion-correlation effect. Moreover, the atomic force microscopy (AFM) images reveal the entire surface is covered with wormlike ss-DNA and the measured surface roughness (1.8±0.2 nm) also matches well with the film thickness by SFA. The desorption behaviors of ss-DNA layer from mica surface occur by adding sodium salt into gap buffer, which is mainly ascribed to the decreased ion-ion correlation force. This paper employing SFA and AFM techniques to characterize the DNA film with flexibility and stable mechanical ability achieved by ion bridging method, is helpful to fabricate the DNA-based devices in nanoscale.