Electrochemical (EC) detection techniques in flow-based analytical systems such as flow injection analysis (FIA), capillary electrophoresis (CE), and liquid chromatography (LC) have attracted continuous interest over the last three decades, leading to significant advances in EC detection of a wide range of analytes in the liquid phase. In this context, the unique advantages of pulsed amperometric detection (PAD) in terms of high sensitivity and selectivity, and electrode cleaning through the application of pulsed potential for noble metal electrodes (e.g. Au, Pt), have established PAD as an important detection technique for a variety of electrochemically active compounds. PAD is especially valuable for analytes not detectable by ultraviolet (UV) photometric detection, such as organic aliphatic compounds and carbohydrates, especially when used with miniaturised capillary and chip-based separation methods. These applications have been accomplished through advances in PAD potential waveform design, as well as through the incorporation of nanomaterials (NMs) employed as microelectrodes in PAD. PAD allows on-line pulsed potential cleaning and coupling with capillary or standard separation techniques. The NMs are largely employed in microelectrodes to speed up mass and electron transfer between electrode surfaces and to perform as reactants in EC analysis. These advances in PAD have improved the sensitive and selective EC detection of analytes, especially in biological samples with complex sample matrices, and detection of electro-inactive compounds such as aliphatic organic compounds (i.e., formic acid, acetic acid, maleic acids, and β-cyclodextrin complexes). This review addresses the fundamentals of PAD, the role of pulsed sequences in AD, the utilisation of different EC detectors for PAD, technological advancements in PAD waveforms, utilisation of microelectrodes in PAD techniques, advances in the use of NMs in PAD, the applications of PAD, and prospects for EC detection, with emphasis on PAD in flow-based systems.