Chitosan has been widely used to prepare a DNA carrier for highly efficient and non-toxic gene therapy. In the present study, we investigated DNA charge neutralization and compaction by chitosan in solutions of various pH levels by dynamic light scattering (DLS), magnetic tweezers (MT), and atomic force microscopy (AFM). We found that when chitosan concentration is higher than a critical value (0.2 µM), corresponding the ratio of phosphate and NH₂ in chitosan k = 1.9 , the electrophoretic mobility of DNA-chitosan complex maintains an almost constant value when pH of solution is less 6.5, the isoelectric point of chitosan. Then it decreases with increasing pH of solution. However, when chitosan concentration is lower than the critical value, the mobility of the complex increases with pH in the range of acidity and reaches the maximum when the pH of the solution approaches the isoelectric point of chitosan. It finally decreases with increasing pH in solutions. The corresponding condensing force of the DNA-chitosan complex measured by single molecular MT changes accordingly with its charge neutralization in the same solution concentration (20 µM) and is consistent with the DLS measurements. This phenomenon might be related to the weakening interaction between DNA and chitosan in low pH solutions, and is verified by measuring the ratio of free chitosan to DNA complex in solutions. We also observed the various morphologies of DNA-chitosan complexes, such as ring, rod, flower, braid, and other structures, under different degrees of deacetylation, molecular weight, solution concentration and pH in solutions by AFM.