A novel approach to enhance the electrochemical sensitivity of screen-printed graphene electrode (SPGE) to dopamine (DA) and uric acid (UA) utilizing sodium dodecylbenzene sulfonate (SDBS) functionalized polypyrrole (PPy) is presented. The functionalized PPy nanosheets (SDBS/PPy) were synthesized via low-temperature polymerization. The introduced dodecylbenzene sulfonate (DBS-) facilitated the dispersion of functionalized PPy nanosheets, resulting in a notably increased specific surface area. DA and UA adsorption behavior on PPy and SDBS/PPy surface was investigated using density functional theory (DFT). The adsorption energy of DA and UA on SDBS/PPy was 0.15 eV and 0.05 eV higher than that on PPy. The adsorption ability of DA and UA was greatly improved after doping DBS- in PPy. The electrochemical response of SDBS/PPy-modified SPGE (SDBS/PPy/SPGE) to different concentrations of DA and UA was evaluated. Compared with SPGE, the oxidation current sensitivity of SDBS/PPy/SPGE to DA increased from 0.08 μA·μM−1 to 0.13 μA·μM−1, the oxidation current sensitivity of SDBS/PPy/SPGE to UA increased from 0.01 μA·μM−1 to 0.04 μA·μM−1. Electrochemical measurement demonstrated that PPy doping with DBS- caused a unique improvement in DA and UA performance of SPGE.