Lung cancer bone metastases usually involve multiple interactions between cancer cells and host organs. To mimic such a structure, a cell co-culture micropattern of A549 lung cancer cells and osteoblast cells (A549/OB) was developed using a μ-eraser strategy for anti-cancer drug evaluation and also understanding cell–cell communication. When applying the μ-eraser strategy, a PDMS stamp was pressed to induce cell lysis. Hence, the minimum pressure required to induce cell lysis was first quantified. Then the pressure and pressing time for the micropatterning process were optimized to obtain cell micropatterns of high fidelity and repeatability. For different types of cells (A549 and human mesenchymal stem cells) and different substrates (TCP and PLGA nanofiber sheets), the optimized pressure was also different (i.e., for A549 on TCP, a pressure of 24.5 kPa was pressed for 10 s). According to a live/dead assay and Alamar Blue assay, cell viability and proliferation potential were not affected by the micropatterning process. There were several advantages for the μ-eraser strategy: substrates were not pre-treated; cell micropatterns mainly relied on the pattern of the PDMS stamp along with the micropatterning process; cells in the micropattern were not restricted in specified regions. Thus the A549/OB co-culture micropattern on TCP was used to evaluate the efficacy of a therapeutic agent (doxorubicin). In the co-culture micropattern, the efficacy of doxorubicin decreased when the expression of ALP in OB was elevated. The co-culture micropattern model showed the potential to be used for new anti-cancer drug development.