Serotonin (5-hydroxytryptamine, 5-HT) plays a crucial role as a monoamine neurotransmitter, regulating various behavioral and physiological functions in the brain and peripheral systems. Its effects encompass emotions, behaviors, gastrointestinal motility, hemostasis, and cardiovascular function. Dysregulation of the serotonergic system and imbalances in 5-HT levels have been associated with psychiatric disorders, underscoring its potential as a biomarker for conditions like anxiety disorders, depression, Alzheimer's disease, and impulsive aggressiveness. However, the precise mechanisms by which 5-HT modulates these physiological conditions and behavioral processes remain unknown, necessitating the use of sensing tools to monitor 5-HT dynamics in specific locations. Traditional techniques such as high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) have been employed to measure 5-HT concentrations in biological samples. However, these offline methods only provide information at the end of an experiment and lack spatial and temporal resolution. Due to the rapid extracellular release and uptake of 5-HT, there is a clear need for detection techniques with high spatiotemporal resolution to investigate serotonergic modulations.This dissertation focuses on the development of minimally invasive neurochemical sensing systems to address challenges related to real-time 5-HT sensing and facilitate in vitro and in vivo investigation of serotonergic modulation. Two sensing systems were developed. For in vitro 5-HT sensing, surface-modified microelectrodes with single carbon fiber were developed and integrated with a portable potentiostat for point-of-care (POC) applications. These microelectrodes were tested for detecting in vitro cell-secreted 5-HT and 5-HT in homogenized crayfish nerve cord samples. The portable system exhibited a sensitivity of 74 nM/µM with a limit of detection (LOD) of 140 nM. Moreover, it was tested for detecting 5-HT in artificial urine, showcasing its application as a POC device for early diagnosis of 5-HT syndrome from urine tests. For in vivo 5-HT sensing, surface-modified microelectrodes with multiple carbon fibers were developed to enhance mechanical robustness specifically for in vivo applications. After integration with a miniature PCB, the device was able to co-detect dopamine (DA) and 5-HT at sub-micromolar concentrations with wireless communication. The integrated untethered implantable system demonstrated its capabilities for in vivo simultaneous monitoring of DA and 5-HT in freely moving crayfish during injection events. Overall, these developed systems offer electrochemical 5-HT sensing solutions for both in vitro and in vivo applications, providing reliable tools to obtain real-time 5-HT dynamics information with high spatial resolution. This capability significantly enhances our ability to investigate precise 5-HT signaling and mechanism underlying serotonergic modulation in the disorder development and behavioral processes.