A novel electrochemical sensor for isoprenaline (IP) comprising a β-Cyclodextrin (β-CD) functionalized graphene oxide (GO) and an electrochemically generated acid yellow 9 polymer as a composite material modified glassy carbon electrode (GCE) has been developed. The composite material was characterized using infrared spectroscopy and scanning electron microscopy. The functionalization of GO with β-CD was scrutinized by varying the content of the β-CD material. Interestingly, the synergistic electrocatalytic activity was examined at different β-CD loadings functionalized GO in the sensitivity for the detection of IP. The electrochemical characterization of the proposed sensor (β-CD-GO/PAY/GCE) was performed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The developed composite based electrode displayed superior sensitivity towards IP over that of the other modified electrodes. The limit of detection at β-CD-GO/PAY/GCE was found to be 3.3 × 10^− 8 M. Moreover, the detection potential of IP was notably lower at the proposed composite based sensor. The β-CD-GO/PAY/GCE was also successfully applied for simultaneous resolution of IP in the presence of endogenous interfering biomolecule such as uric acid. The recovery results attained for real samples recommend practical utility of the proposed composite electrode an effective and reliable electrochemical sensor for quantification of IP. The sensor is highly stable, reproducible and signifies a feasible platform for the analysis of IP.