The electrochemical behavior of ciprofloxacin (CFX) and its interaction with the natural calf thymus DNA (ctDNA) is studied by using pulse difference voltammetry on a carbon electrode. CFX shows a well-defined oxidative peak at+0.88 V. As a result of reaction with ctDNA,the oxidative peak of CFX decreased markedly. According to the electrochemical equation deduced in this paper, the binding constant of 1.36 × 105 (mol/L)-1 and the binding size of 1.94 (base pairs) of CFX with ctDNA were obtained by nonlinear fit analysis of the electrochemical data. The mechanism of the interaction was explored.