To effectively monitor human performance biomarkers, we applied electrokinetic preconcentration technique of the biomarkers onto patterned graphene-modified\ electrodes in a nanochannel by frequency-selective dielectrophoresis using the molecular dam operation. Low copy numbers of human performance biomarkers can be enriched several orders of magnitudes and detected in tens of seconds. Employing this methodology and electrochemical detection, we have demonstrated the ultrafast and ultrasensitive detection of human performance biomarkers, such as neuropeptide NPY and OXA at pM levels, cortisol at detection limit of 30 pg/ml, all can be detected in 5 minutes, in addition to cancer biomarker prostate specific antigen at 1 pg/ml in 30 seconds. All these are achieved from subnanoliter samples, with sufficient signal sensitivity to avoid from high levels of interferences within biological matrices. We obtained kinetic curves for titration of various target concentrations, developed testing nanoslit devices for spiked samples, and tested nanoslit devices for clinically relevant samples with nanoscale DEP molecular dam target enrichment or capturing. We advanced the field by first ever applications of nanoscale dielectrophoretic molecular dam as sample enrichment technique combining grapheme-modified electrode for electrochemical detection for ultrafast and ultrasensitive human performance biomarkers monitoring. Given the high sensitivity of the methodology within small volume samples, we envision its utility toward off-line detection from droplets collected by microdialysis for the eventual measurement of human performance biomarkers at high spatial and temporal resolutions.