The complexity of biological fluids makes the sample preparation necessary in the analytical process. The traditional techniques, such as liquid-liquid extraction and solid-phase extraction, are time-consuming and labor-intensive. Turbulent flow chromatography (TFC), as an on-line direct injection technique, possesses the advantages of reducing sample preparation steps and enabling the effective pre-concentration and clean-up of biological fluids. Therefore, it is a high-throughput and high-selectivity technique for biological sample pretreatment. In this review, the theory, advantages, characteristics and current status of TFC in bioanalysis are introduced and summarized.