Small interfering RNA (siRNA) screening approaches have provided useful tools for the validation of genetic functions; however, image-based siRNA screening using multiwell plates requires large numbers of cells and time, which could be the barrier in application for gene mechanisms study using human adult cells. Therefore, we developed the advanced method with the cell-defined siRNA microarray (CDSM), for functional analysis of genes in small scale within slide glass using human bone marrow stromal cells (hBMSCs). We designed cell spot system with biomaterials (sucrose, gelatin, poly-l-lysine and matrigel) to control the attachment of hBMSCs inside spot area on three-dimensional (3D) hydrogel-coated slides. The p65 expression was used as a validation standard which described our previous report. For the optimization of siRNA mixture, first, we detected five kinds of commercialized reagent (Lipofectamine 2000, RNAi-Max, Metafectine, Metafectine Pro, TurboFectin 8.0) via validation. Then, according to quantification of p65 expression, we selected 2 μl of RNAi-Max as the most effective reagent condition on our system. Using same validation standard, we optimized sucrose and gelatin concentration (80 mM and 0.13%), respectively. Next, we performed titration of siRNA quantity (2.66–5.55 μM) by reverse transfection time (24 h, 48 h, 72 h) and confirmed 3.75 μM siRNA concentration and 48 h as the best condition. To sum up the process for optimized CDSM, 3 μl of 20 μM siRNA (3.75 μM) was transferred to the 384-well V-bottom plate containing 2 μl of dH2O and 2 μl of 0.6 M sucrose (80 mM). Then, 2 μl of RNAi-Max was added and incubated for 20 min at room temperature after mixing gently and centrifugation shortly. Five microliters of gelatin (0.26%) and 2 μl of growth factor reduced phenol red-free matrigel (12.5%) were added and mixed by pipetting gently. Finally, optimized siRNA mixture was printed on 3D hydrogel-coated slides and cell-defined attachment and siRNA reverse transfection were induced. The efficiency of this CDSM was verified using three siRNAs (targeting p65, Slug, and N-cadherin), with persistent gene silencing for 5 days. We obtained the significant and reliable data with effective knock-down in our condition, and suggested our method as the qualitatively improved siRNA microarray screening method for hBMSCs.