Tryptophan and melatonin are pleiotropic molecules, each capable of influencing several cellular, biochemical, and physiological responses. Therefore, sensitive detection of tryptophan and melatonin in pharmaceutical and human samples is crucial for human wellbeing. Mass spectrometry, high-performance liquid chromatography, and capillary electrophoresis are common methods for both tryptophan and melatonin analysis; however, these methods require copious amounts of time, money, and manpower. Novel electrochemical and optical detection tools have been subjects of intensive research due to their ability to offer a better signal-to-noise ratio, high specificity, ultra-sensitivity, and wide dynamic range. Recently, researchers have designed sensitive and selective electrochemical and optical platforms by using new surface modifications, microfabrication techniques, and the decoration of diverse nanomaterials with unique properties for the detection of tryptophan and melatonin. However, there is a scarcity of review articles addressing the recent developments in the electrochemical and optical detection of tryptophan and melatonin. Here, we provide a critical and objective review of high-sensitivity tryptophan and melatonin sensors that have been developed over the past six years (2015 onwards). We review the principles, performance, and limitations of these sensors. We also address critical aspects of sensitivity and selectivity, limit and range of detection, fabrication process and time, durability, and biocompatibility. Finally, we discuss challenges related to tryptophan and melatonin detection and present future outlooks.