Carbon is the material of choice for electroanalysis of biological systems, being particularly applicable to neurotransmitter analysis as carbon fiber microelectrodes (CFMs). CFMs are most often applied to dopamine detection; however, the scope of CFM analysis has rapidly expanded over the last decade with our laboratory's focus being on improving serotonin detection at CFMs, which we achieved in the past via Nafion modification. We began this present work by seeking to optimize this modification to gain increased analytical sensitivity toward serotonin under the assumption that exposure of bare carbon to the in vivo environment rapidly deteriorates analytical performance. However, we were unable to experimentally verify this assumption and found that electrodes that had been exposed to the in vivo environment were more sensitive to evoked and ambient dopamine. We hypothesized that high in vivo concentrations of ambient extracellular glutamate could polymerize with a negative charge onto CFMs and facilitate response to dopamine. We verified this polymerization electrochemically and characterized the mechanisms of deposition with micro- and nano-imaging. Importantly, we identified that the application of 1.3 V as a positive upper waveform limit is a crucial factor for facilitating glutamate polymerization, thus improving analytical performance. Critically, information gained from these dopamine studies were extended to an in vivo environment where a 2-fold increase in sensitivity to evoked serotonin was achieved. Thus, we present here the novel finding that innate aspects of the in vivo environment are auspicious for detection of dopamine and serotonin at carbon fibers, offering a solution to our goal of an improved fast-scan cyclic voltammetry serotonin detection paradigm.