In order to achieve the aim of this study, i.e., simultaneous measurement of trace amounts of dopamine (DA) and uric acid (UA) following their preconcentration, a novel sorbent with high performance was prepared. Hence, herein, poly vinyl chloride/Mg-Al layered double hydroxide (PVC/Mg-Al LDH) electrospun nanofibers were synthesized for the thin film- micro solid phase extraction (TF-µSPE) and then, simultaneous extraction of trace amounts of DA and UA was done. Finally, DA and UA were determined simultaneously from two different human plasma samples; afterwards, glassy carbon electrode/Poly pyrrole/diethylamine (DA, UA) molecularly- imprinted polymer (GCE/Ppy/DEA (DA, UA) MIP) electrochemical biosensor was constructed. The synthesis confirmation of PVC-Mg-Al LDH electrospun nanofibers was accomplished by scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, X-ray diffraction (XRD) and attenuated total reflection-Fourier transform infrared (ATR-FTIR). In addition, the GCE/Ppy/DEA MIP and GCE/Ppy/DEA (non-imprinted polymer) NIP nanocomposites were characterized with field emission-scanning electron microscopy (FE-SEM). PVC, as a polymer with a large surface area and high porosity, is an appropriate candidate in the sorbent preparation process with electrospinning. The flexible interlayer region is accessible to various anionic and polar molecular species. Adding Mg-Al LDH led to an extensive surface area of electrospun nanofibers. In the case of Ppy and DEA MIP as GCE surface modifiers, Ppy increased the electroactive sites, leading to the high conductivity of the electrochemical biosensor. Besides, DEA MIP has high selectivity and sensitivity. Optimization of impressive factors on the extraction procedure was conducted by one at-time (OAT) method, and optimum conditions for the construction of electrochemical biosensor were attained with experimental design (design-expert 12.0 software). According to the optimum conditions, the wide linear range was obtained 76.6–268.1 µg mL−1 for DA and 84.1–378.2 µg mL−1 for UA with R2≥ 0.96, low detection limits ranged from 22.3 µg mL–1 for DA and 24.8 µg mL−1 for UA based on S/N = 3 and large enrichment factors. The intra-day (n = 3) and inter-day (n = 3) RSDs% were obtained in the span of 5.3% − 6.1% for DA and 4.0% − 5.6% for UA. Eventually, the efficiency of the TF-µSPE-voltammetric biosensor method was evaluated for the measuring of DA and UA in human plasma samples with satisfactory recoveries (90.2%–99.7%).