We review the state of the art in microfluidic separation technique based two-phase laminar flow with an application focus on chemical and biological sample. As we describe herein, two-phase laminar flow in the microfluidic extraction has several biological and engineering advantages over other methods including high reproducibility, biocompatibility, and selectivity. We review advances in applications of two-phase laminar flow and examine key parameters such as flow rate, phase composition, and surface charge property, how these can affect extract performance with the technology including microfluidic separation system. A special technology focus is given to emerging novel integrative microfluidic extraction, which aims to merge aqueous phase laminar flow and electric field technologies into simple packages. We conclude with a brief discussion of some of the emerging challenges in the field and some of the approaches that are likely to enhance their application.