A nanocurcumin (NCur)-VO2+ ensemble–based optical nanoprobe is proposed for monitoring of human serum albumin (HSA) and transferrin (TF) in biofluids of serum and urine. The determination strategy of HSA and TF is based on the decrease of the absorbance/color intensity of NCur in the presence of VO2+ due to the formation of NCur-VO2+ ensemble. This leads to aggregation of the NCur and the color change of solution from orange to pale pink. Upon addition of HSA or TF, release of VO2+ from NCur-VO2+ ensemble occurs due to their stronger binding affinities to VO2+ in competition with the NCur. This leads to deaggregation of the NCur and recovery of the decreased absorption/color intensity within a defined time range. The absorption changes at λ = 455 and the color of NCur solution can be monitored spectrophotometrically or visually by a smartphone camera, respectively. Under optimal conditions, the analytical signals increase linearly in the ranges 50–200 nM (LOD = 11 nM) and 20–140 nM (LOD = 8 nM) for HSA and TF, respectively. The difference in the different affinities between the HSA and the TF for binding to VO2+ produces the unique time profiles of each protein. Therefore, the simultaneous determination of HSA and TF is provided by using the least-square support-vector machine (LS-SVM) model. The good recoveries and small errors of predicted values suggest that the nanoprobe is capable to resolve binary mixtures of HSA and TF. The method was applied to the simultaneous determination of HSA and TF in serum and urine samples.