A new method to integrate poly-dl-lactide (PLA) patterned electrospun fibers with a polydimethylsiloxane (PDMS) microfluidic chip was successfully developed via lithography. Hepatocyte behavior under static and dynamic conditions was investigated. Immunohistochemical analyses indicated good hepatocyte survival under the dynamic culture system with effective hepatocyte spheroid formation in the patterned microfluidic chip vs. static culture conditions and tissue culture plate (TCP). In particular, hepatocytes seeded in this microfluidic chip under a flow rate of 10 μL/min could re-establish hepatocyte polarity to support biliary excretion and were able to maintain high levels of albumin and urea secretion over 15 days. Furthermore, the optimized system could produce sensitive and consistent responses to nano-Ag-induced hepatotoxicity during culture. Thus, this microfluidic chip device provides a new means of fabricating complex liver tissue-engineered scaffolds, and may be of considerable utility in the toxicity screening of nanoparticles.