Although thousands of in vitro selection and evolution experiments have been performed to seek different types of targets, most of them have only inspected the terminal evolutionary pool for patterns. In addition, to rapidly obtain the most favorable target, many experiments have been carried out under increasing selection pressure. However, increasing selection pressure seldom occurs in natural evolution. We studied the dynamic features of DNA in vitro evolution in the presence of the Mnt repressor under sequential constant selection pressure. When evolving under a constant pressure from an initial random pool of DNA, our system showed a clear, sharp, and reproducible crossover from a random population to an advantageous population (higher binding affinities of DNA sequences to the Mnt repressor). This crossover occurs after a long latent period during which there are no obvious changes in the population phenotype. We demonstrated that the existence of the crossover is caused by a significant sequence-nonspecific binding in the repressor–DNA system. After the crossover, the population settled in a stationary distribution of genotypes, which responded immediately to a subsequent sudden increase in selection pressure. We also experimentally tested the linear correlation between the evolution speed and sequence diversity (Fisher’s theorem) in our system.