Bioanalysis usually requires a preparation procedure for sample cleanup or preconcentration. Conventional sample preparation techniques are often time consuming and labor intensive. Among recent progress in sample preparation, single drop microextraction (SDME) is one of the most efficient techniques providing both sample cleanup and preconcentration capabilities. In SDME, analytes are extracted from a sample solution into an acceptor drop and the drop is introduced to subsequent analysis. Since the volume of the acceptor drop is 1-10 µl or less, the consumption of solvents can be minimized and the preconcentration effect is enhanced. In this review, the basic principles of two-phase and three-phase SDME are described briefly and then recently developed modes of SDME, coupling with analytical instruments, and methods to enhance the drop stability are discussed. Recent applications of SDME to biological samples, including urine, blood and saliva, for the analysis of drugs, metal ions and biomarkers are reviewed.