Winter wheat leaf and canopy nitrogen status are important indicators for crop growth diagnosis and grain protein content (GPC) estimation. We monitored the N status and GPC of winter wheat based on wheat leaf-level and canopy-level chlorophyll sensitivity parameters using two hand-held instruments: SPAD-502 for wheat leaves and Multiplex 3 for the canopy. The SPAD value of the first two fully expanded leaves of winter wheat and nine wheat canopy fluorescence parameters were measured at five growth stages from April to June 2012. The correlation between the SPAD value and the canopy fluorescence parameters were then analyzed and compared for their accuracy in assessing leaf N content (LNC), canopy N density (CND), and GPC. Canopy-level fluorescence parameters were more accurate for estimating CND than leaf SPAD and LNC. The results of our study also indicate that leaf-level SPAD and canopy-level fluorescence spectral parameters are all sensitive to GPC. A partial least-squares regression (PLSR) method was used to estimate GPC based on the multi-temporal leaf SPAD value and canopy fluorescence parameters. Estimating GPC using PLSR with multiple stages of fluorescence variables yielded greater estimation accuracy [R²= 0.766 and root mean square of error prediction (RMSEP) = 0.563%] than the SPAD variables [R²= 0.653 and RMSEP = 0.707%]. Thus, we conclude that canopy-level fluorescence spectral parameters are better indicators for wheat growth activity and can be used to successfully estimate winter wheat GPC.