Background and Purpose It is essential to develop a reliable predictive serum biomarker for Parkinson’s disease (PD). The accumulation of alpha-synuclein (αSyn) and up-regulated expression of Rab35 participate in the etiology of PD. The purpose of this investigation was to determine whether the combined assessment of serum αSyn and Rab35 is a useful predictive biomarker for PD. Methods Serum levels of αSyn or Rab35 were determined in serum samples from 59 sporadic PD patients, 19 progressive supranuclear palsy (PSP) patients, 20 multiple system atrophy (MSA) patients, and 60 normal controls (NC). Receiver operating characteristics (ROC) curves were calculated to determine the diagnostic accuracy of αSyn or/and Rab35 in discriminating PD patients from NC or atypical parkinsonian patients. Results The levels of αSyn and Rab35 were increased in PD patients. The serum level of Rab35 was positively correlated with that of αSyn in PD patients. Compared to analyzing αSyn or Rab35 alone, the combined analysis of αSyn and Rab35 produced a larger area under the ROC curve and performed better in discriminating PD patients from NC, MSA patients, or PSP patients. When age was dichotomized at 55, 60, 65, or 70 years, the combined assessment of αSyn and Rab35 for classifying PD was better in the group below the cutoff age than in the group above the cutoff age. Conclusions Combined assessment of serum αSyn and Rab35 is a better biomarker for discriminating PD patients from NC or atypical parkinsonian patients, and is a useful predictive biomarker for younger sporadic PD patients.