Chemotherapy remains the primary systemic treatment for patients with neuroblastoma, but the lack of appropriate in vitro tumor microenvironment models has resulted in suboptimal efficacy of chemotherapeutic agents. In this study, we propose innovative bioresponsive hydrogel microfibers that replicate the mechanical properties of extracellular matrix surrounding neuroblastoma cells for assessing tumor drug responses. These microfibers are composed of an alginate/poly (N-Isopropyl acrylamide) shell and a carboxymethyl cellulose core, fabricated by microfluidic technology. Due to the precise manipulation afforded by microfluidics, it is possible to continuously generate fibers that encapsulate cells with uniform size and precise structure. Additionally, the rapid temperature response characteristics enabled the microfibers to mimic the mechanical properties of the extracellular matrix, thereby regulating the cellular pressure environment and rapidly forming highly active three-dimensional tumor spheroids. Ultimately, our findings demonstrate that neuroblastoma spheres within the microfibers display varying sensitivities to different chemotherapy drugs under distinct external pressure conditions. In conclusion, this biomimetic extracellular matrix microfiber offers a dependable foundation for replicating the neuroblastoma microenvironment and facilitating the assessment of clinical drug efficacy.