Carbon nanotubes are a kind of new carbon-based nanomaterials, which have drawn great attention in many application fields including the biomarker analysis in biomedical and clinical research. In the present study, the feasibility of single-walled carbon nanotubes (SWCNTs) as solid-phase extraction (SPE) adsorbent for the determination of amino acid neurotransmitters (AANs) (glutamic, dopamine and γ-amino-n-butyric acid) in human cerebrospinal fluids (CSFs) was investigated. Various parameters affecting SPE efficiency including the eluent and its volume, adsorbent amount and sample volume were systematically studied. The acquired calibration curves were linear (r2 > 0.999) over the concentration range 10 to 500 ng per injection. By using SWCNTs as the SPE adsorbent, the detection limit of 12.9-42.5 fmol (at S/N of 3) were achieved with the preconcentration efficiency of more than 500-folds. The feasibility of the proposed method was validated by successfully applied to the measurements of ANNs in human CSFs sampled from healthy subjects and the patients with human immunodeficiency virus (HIV) infection, malignant melanoma and hypertension. This original application represents a powerful tool to study the kinetics of ANNs release by neuronal cells during neurotransmission, as well as for the understanding of the pathobiological and therapeutic basis of these remarkable molecular for diverse diseases.