A novel approach for DNA sequence preprocessing by merging intelligent detection is proposed. This approach can automatically find and locate contaminants using statistical analysis methods, random search and graph-theoretic operations, while no extra background information, such as vector sequence, splice site and clone adapter are needed during preprocessing. Experiments on Zebrafish DNA show that the approach can significantly improve the efficiency and accuracy of DNA sequence preprocessing and provide more stable performance than the conventional methods do, particularly in high-throughput DNA sequence preprocessing.