Pentaploid strawberry (FxLs1137, 2n=5x=35) derived from the hybridization between diploid Fragaria viridis (male) and octaploid F. × ananassa ‘Fusanoka’ (female), and its allodecaploid (A3, 2n=10x=70) were employed for conducting proteomic analysis with two dimensional gel electrophoresis technology under observation of agronomic traits, soil and plant analyzer development (SPAD) readings, and the net photosynthetic rate. (1) Compared with FxLs1137, A3 showed a significant dwarf type, larger crown, increased leaf length and leaf width, much thicker in leaf, more dark green leaves, and it also has a bigger SPAD for leaf and net photosynthetic rate; (2) Protein spots of FxLs1137 and A3 analyzed using PDQuest software were mainly scattered in the isoelectric point ranging from pH 4 to 7, and molecular weight ranging from 14.4 to 66.2 kD. More than 700 expressed protein spots in FxLs1137 and A3 were detected. Expression levels of 18 protein spots changed over 1.5 fold after chromosome doubling, and 4 protein spots changed over 2.0 fold. We identified these 4 protein spots with MALDITOFMS/MS mass spectrometry technology, and results showed that the 4 proteins are major strawberry allergen Fra a 1E, heterogeneous nuclear ribonucleoprotein 1, lipoyl synthase 1, chloroplastic, and NAD(P)H dehydrogenase (quinine) FQR1like. These proteins are involved in stress resistant, mRNA transport, material and energy metabolism. Results of realtime quantitative PCR of gene encoding the above four proteins demonstrated that the difference tendency of gene transcriptional expression was consistent with the proteins levels. We obtained the differentially expressed proteins in strawberry chromosome doubling, which provided tracks for further research.