Wound healing is a complex process composed of different stages, which involves extensive communication between the different cellular factors of the extracellular matrix (ECM). The radio frequency electromagnetic field (RF-EMF) has been used to accelerate the wound-healing process and it has been found to enhance cell alignment and mobility. The conventional methods for cell mobility analysis in an electromagnetic field generated by a radiation source are not advisable due to the low-precision, nonuniform distribution of the field, low efficiency of the analysis in batch and the lack of system integration for autonomous on-body operation. Here, a novel and versatile electromagnetic exposure system integrated with a microfluidic chip was fabricated to explore the EMF-induced response. A gradient electromagnetic field in a two-dimensional plane has been successfully established in the microchambers placed along the field line. In this work, by deploying our radiation experiments in vitro, we validated the on-chip monitoring of cell response to exposure. This electromagnetic field was simulated and human amniotic epithelial cells (HAECs) were cultured in different microchambers for continuous exposure to the electromagnetic field excited by a monopole RF antenna (1.8 GHz). New protrusions were generated and an obvious increase in filopodia with the increased field intensity was investigated. Meanwhile, the variation in intracellular Ca2+ concentration under the electromagnetic field was examined. The inhibitory effect of the Ca2+ circulation was further inspected to reveal the potential downstream signaling pathway in the RF-EMF-related bioassay, suggesting that cytoskeletal dynamics of cells under exposure are highly associated with the EGF receptor (EGFR)-cytoskeleton downstream signaling pathway. Finally, the field-induced cell elongation and alignment parallel to the field direction were observed. Additionally, the subsequent recovery (field withdrawal) and re-establishment (field re-exposure) were explored. These results indicated that this reliable and versatile exposure system for bioassay could achieve precise and high-throughput detection of the RF-EMF-induced cytoskeletal reorganization in vitro and evaluate the possible health risk from RF-EMF exposure.