The binary nanomaterials and graphitic carbon based hybrid has been developed as an important porous nanomaterial for fabricating electrode with applications in non-enzymatic (bio) sensors. We report a fast synthesis of bimetal oxide particles of nano-sized manganese ferrite (MnFe2O4) decorated on graphitic carbon nitride (GCN) via a high-intensity ultrasonic irradiation method for C (30 kHz and 70 W/cm2). The nanocomposites were analyzed by powder X-ray diffraction, XPS, EDS, TEM to ascertain the effects of synthesis parameters on structure, and morphology. The MnFe2O4/GCN modified electrode demonstrated superior electrocatalytic activity toward the neurotransmitter (5-hydroxytryptamine) detection with a high peak intensity at +0.21 V. The appealing application of the MnFe2O4/GCN/GCE as neurotransmitter sensors is presented and a possible sensing mechanism is analyzed. The constructed electrochemical sensor for the detection of 5-hydroxytryptamine (STN) showed a wide working range (0.1–522.6 μM), high sensitivity (19.377 μA μM−1 cm−2), and nano-molar detection limit (3.1 nM). Moreover, it is worth noting that the MnFe2O4/GCN not only enhanced activity and also promoted the electron transfer rate towards STN detection. The proposed sensor was analyzed for its real-time applications to the detection of STN in rat brain serum, and human blood serum in good satisfactory results was obtained. The results showed promising reproducibility, repeatability, and high stability for neurotransmitter detection in biological samples.