Background The recovery of upper extremity motor deficits represents a functional challenge for persons with stroke. The aim of this study was to evaluate the mechanisms of Unilateral task-oriented training combined with dynamic hand orthosis for the recovery in subacute stroke by using functional near-infrared spectroscopy (fNIRS). Methods This was a pilot randomized, controlled, assessor-blinded trial. A total sample of 30 subacute persons with stroke were randomized into two groups: the orthosis group and the usual exercise group. All participants received four weeks (60 min/day, 5 days/week) of unilateral upper limb training. The orthosis group wore a dynamic hand orthosis during half the training (30 min/day). O2Hb concentrations and a laterality index for bilateral premotor cortex, sensorimotor cortex by fNIRS were calculated as outcome indicators that were measured at baseline and 4 weeks after the interventions. Clinical outcomes were collected for correlation analysis. Results No significant difference in O2Hb concentrations change between two groups. Cortical activation shifted from the ipsilateral to the contralateral premotor cortex and from the contralateral to the ipsilateral sensorimotor cortex in both groups. In the Orthosis group, the change of Fugl-Meyer Assessment of arm score was positively correlated to the shift to contralateral premotor cortex activation and the change of Action Research Arm Test score was positively correlated to the shift to ipsilateral sensorimotor cortex activation. In the UL group, a negative correlation was observed between the shift to ipsilateral sensorimotor cortex and changes of Fugl-Meyer Assessment of arm score. Conclusion This is the first fNIRS study to explore the mechanism of recovery in subacute stroke after utilizing unilateral task-oriented training and a dynamic orthosis. The current results suggest that fNIRS combined with clinical tests may be useful to evaluate the mechanism of stroke recovery after different rehabilitation interventions. Trial registration: Registered on January 11, 2018 at ClinicalTrials.gov (NCT03396939). https://clinicaltrials.gov/ct2/home.