Listeria monocytogenes (LMs) that release 3-hydroxy-2-butanone (3H-2B) biomarker, can cause serious human illness and even death; in this regard, conventional techniques for the smart detection of LMs are of vital importance; however, they still suffer from the requirement of either sophisticated techniques or complicated instrumentation. Herein, we report a metal-oxide semiconductor (MOS) sensor, built with 3D mesoporous nickel oxide nanocuboids (M-NiO NCs), for the detection of LMs. The M-NiO NCs were obtained by directly sacrificing nickel foam in oxalic acid, followed by subsequent annealing. The M-NiO NCs have a rough surface and many multichannel pathways. Impressively, gas sensors built with M-NiO NCs display excellent sensing performance towards the 3H-2B biomarker, exhibiting an ultrahigh sensitivity (R_g/R_a = 302 at 50 ppm) and excellent selectivity. Moreover, the 3H-2B sensor presents excellent long-term stability and, particularly, a low limit of detection (LOD = 0.5 ppm) at 120 °C. The multichannel pathways of the M-NiO NCs contribute to gas adsorption and diffusion, thus enhancing the sensing behavior. Our study provides an ingenious and low-cost strategy to produce mesoporous NiO nanostructures as well as new insights for the detection of pathogenic microbes in food.