Histamine is an organic nitrogenous compound released from mast cells, often as part of an instant immune response. Life-threatening anaphylaxis can occur with significant histamine intolerance, which is also referred to as histaminosis caused by overproduction of histamine in the body. Herein, we demonstrate the synthesis of FeS2-incorporated rGO aerogel (FeS2-AG) by lyophilization, as an active SERS substrate for the detection of histamine in blood serum. The morphological characterization revealed the nanoflower structure of FeS2 and uniform porosity of the aerogel, in which the nanoflower structure of FeS2 was homogenously distributed on the surface of the porous aerogel. The cubic structure of FeS2 nanoflowers was revealed by X-ray diffraction and Fourier transform infrared spectroscopy demonstrated the characteristic peak at ∼1176 cm−1 which is related to pyrite surface chemistry of FeS2 nanoparticles. The as-fabricated sensor exhibited an enhancement factor of 1.61 × 107 with a limit of detection of 0.08 ng mL−1. The substrate exhibited a good selectivity and linear range of detection towards histamine in the presence of 2-fold concentration of ascorbic acid, glucose, urea, and uric acid. This excellent performance of the sensor can be ascribed to the chemical enhancement occurring via the interaction of oxy functional groups of aerogel and multiple coordination sites (ferrous ions (Fe2+)) of FeS2 with histamine molecules causing reconstruction of the band gap. This newly formed energy band further leads to the occurrence of the Raman resonance effect, providing an enhancement in the SERS signal over that of the Raman signal obtained from bulk histamine. The sensor showed good recovery percentage of 97.25% from real samples, highlighting the efficiency of the as-fabricated sensor towards histamine detection. © 2023 The Royal Society of Chemistry.