The root system of plants is a major component of their bodies in terms of both function and bulk. The investigation of root system development is greatly assisted by microfluidic devices, which improve the spatial and temporal resolution of observations without destroying tissue. In the present study, a multi-chamber petaloid root-growth chip was developed for studying the development and physiology of root systems that have thin branching structures (i.e., fibrous root systems). The petaloid root-growth chip includes a central seed germination chamber and five root-growth chambers for observing the development of fibrous roots. The proposed device was applied for investigating the root system development of Oryza sativa. The phenotype and growth kinetics of O. sativa root systems grown in the proposed device were compared with those obtained during growth in a conventional conical flask with agar-based medium, and the results indicated that cultivation in the miniaturized device did not delay root system growth in the early stage (≤2 weeks). In addition, the transparent device enabled the non-destructive observation of the developmental and microstructural characteristics of the roots, such as the root caps, root border cells, and root hairs. Moreover, the ability to control the microenvironment in each of the five root-growth chambers individually facilitated the investigation of specific adaptations in the fibrous root growth of single O. sativa seedlings to different drought stresses. Accordingly, five polyethylene glycol (PEG)6000-induced drought stress conditions were established in the five root-growth chambers to investigate the root development of a single O. sativa seedling in the central germination chamber. In situ observations demonstrated that the different PEG6000-induced conditions affected the root growth responses and root microstructural adaptations of the single seedlings in each root-growth chamber. Therefore, the petaloid root-growth microfluidic chip can eliminate the effects of variations in different plant seeds to reveal the responses of plants to different environmental conditions more objectively while concurrently allowing for non-destructive observations at very high spatial and temporal resolution.