Aflatoxins-B1 (AFB1) and Ochratoxin-A (OchA) are the two types of major mycotoxin produced by Aspergillus flavus, Aspergillus parasiticus fungi, Aspergillus carbonarius, Aspergillus niger, and Penicillium verrocusumv. These toxins are mainly found in metabolite cereals, corn, coffee beans, and other oil-containing food items. Excessive consumption of these toxins can be carcinogenic and lead to cancer. Thus, their rapid testing became essential for food quality control. Herein, manganese oxide nanoparticles (MnO2 nps) have been proposed to explore the interaction with AFB1 and OchA using UV-visible spectroscopy. MnO2 nps were synthesized using the co-precipitation method. They were pure and crystalline with an average crystallite size of 5–6 nm. In the UV-vis study, the maximum absorbance for MnO2 nps was observed around 260 nm. The maximum absorbance for AFB1 and OchA was observed at 365 and 380 nm, respectively, and its intensity enhanced with the addition of MnO2 nps. Sequential changes were observed with varying the concentration of AFB1 and OchA with a fixed concentration of MnO2 nps, resulting in proper interaction. The binding constant (kb) and Gibbs free energy for MnO2 nps-AFB1 and OchA were observed as 1.62 × 104 L g−1 and 2.67 × 104 L g−1, and −24.002 and −25.256 kJ/mol, respectively. The limit of detection for AFB1 and OchA was measured as 4.08 and 10.84 ng/ml, respectively. This bio‐active free direct sensing approach of AFB1 and OchA sensing can be promoted as a potential analytical tool to estimate food quality rapidly and affordable manner at the point of use.