A new green method for the synthesis of poly(sodium 4-styrenesulfonate) functionalized graphene (PSS-graphene) in aqueous solution was realized by using graphene oxide nanosheets (GO) functionalized with PSS and ascorbic acid (AA) as an reducing agent. Various techniques, including Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy and electrochemistry have been utilized to characterize the as-made nanocomposites. The PSS-graphene was used as a modifier to fabricate an electrochemical sensor of tryptophan (Trp). Due to the difference of the adsorption capacity of PSS-graphene for Trp and Tyr, as well as the different charge states of Trp and the other interfering substances, the sensor showed excellent selectivity for Trp. The synergistic amplification of the stronger enrichment and the enhanced electron transfer rate, the signal of Trp on the PSS-graphene modified electrode was about 100-fold enhanced compared to that of the bare GCE. The peak current of Trp is proportional to its concentration in a wide range from 0.04 to 10.0 µmol L with detection limit of 0.02 µmol L. Further, the proposed sensor was applied for the detection of Trp in human serum samples. Favorable results revealed that this work provided a new design strategy and a valuable platform for highly selective and sensitive determination of Trp in complex samples.