Single atom catalysts have been recognized as potential catalysts to fabricate electrochemical biosensors, due to their unexpected catalytic selectivity and activity. Here, we designed and fabricated an ultrasensitive dopamine (DA) sensor based on the flower-like MoS₂ embellished with single Ni site catalyst (Ni-MoS₂). The limit of detection could achieve 1 pM in phosphate buffer solution (PBS, pH = 7.4), 1 pM in bovine serum (pH = 7.4), and 100 pM in artificial urine (pH = 6.8). The excellent sensing performance was attributed to the Ni single atom axial anchoring on the Mo atom in the MoS₂ basal plane with the Ni-S₃ structure. Both the experiment and density functional theory (DFT) results certify that this structural feature is more favorable for the adsorption and electron transfer of DA on Ni atoms. The high proportion of Ni active sites on MoS₂ basal plane effectively enhanced the intrinsic electronic conductivity and electrochemical activity toward DA. The successful establishment of this sensor gives a new guide to expand the field of single atom catalyst in the application of biosensors.