Vanadium-doped porous Co3O4 (V-porous Co3O4) was synthesized via a simple soft-templating method and used as a superior peroxidase mimic for the simultaneous colorimetric determination of glucose and total cholesterol (TC) in whole blood samples on a two-dimensional microfluidic paper-based analytical device (2D-μPAD). The large surface area and the presence of two metals in V-porous Co3O4 contributed to its excellent catalytic activity toward 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 3,3',5,5'- tetramethylbenzidine (TMB) with Michaelis-Menten constants (KM) of 0.1301 and 0.0141 mM, respectively. The 2D-μPAD was fabricated using simple wax screen-printing and cutting techniques. The colorimetric reactions of both glucose and TC on 2D-μPAD were simultaneously performed by adding a single drop of a whole blood sample on the sample zone made of the LF1 membrane. After the enzymatic reactions, the generated hydrogen peroxide (H2O2) was oxidized by V-porous Co3O4 to produce hydroxy radicals (•OH), inducing ABTS and TMB to generate colored products. The generated H2O2 was proportional to the intensities of the green and blue products of the glucose and TC systems, respectively. The developed 2D-μPAD required a short analysis time (∼5 min) with small volumes of samples (15 μL of whole blood) whereby no sample preparation was needed. Owing to several advantages including simplicity, low cost, long-term stability, and simultaneous readout, the novel V-porous Co3O4 coupled with 2D-μPAD proved to be promising for practical uses as a pioneering portable device for the determination of glucose, TC, and other important biomarkers without the need of technical supports.