Artificial sweeteners (ASs) are extensively used as food additives in drinks and beverages to lower calorie intake and prevent lifestyle diseases such as obesity. Although clinical and epidemiological data revealed the link between the chronic overconsumption of ASs and adverse health effects, there still exist controversies over the potential adverse neural toxic effect of ASs such as aspartame (APM), with acceptable daily intake (ADI) for a long time, on human health. In addition, whether APM and its metabolites are neurotoxic remains debatable due to a lack of data from an animal experiment or clinical investigation. Herein, to fully describe the potential neurological effect of APM, adult zebrafish served as the animal model to assess neurophysiological alteration induced by APM exposure within the range of the ADI (1, 10, and 100 mg/L) for 2 months. A cohort of standardized neurobehavioral phenotyping assays was conducted, including light/dark preference tests (LDP), novel tank diving tests, novel object recognition tests, social interaction tests, and color preference tests. For instance, in the LDP test, saccharin remarkably decreased the swimming time of zebrafish in the DARK part from 111 ± 10.8 (control group) to 72.2 ± 11.4 (100 mg/L groups). Besides, brain chemistry involved in the alteration of total neurotransmitters was determined by LC−MS/MS to confirm the behavioral results. Overall, current research studies revealed that APM within the range of the ADI altered the total behavioral profiles of zebrafish and disturbed the homeostasis of neurotransmitters in the brain. The present study has established a set of experimental paradigms, revealing the standardized procedure of using adult zebrafish to determine the neural activity or toxicity of AS molecules phenotypically. Zebrafish behavioral phenotyping methods, which were characterized by a cohort of behavioral fingerprints, can link the phenotypical alteration to changes in neurotransmitters in the brain, so as to provide a predictive reference for the further exploration of the molecular mechanism of phenotypic changes induced by ASs.