Microfluidics-assisted spatially barcoded microarray technology offers a high-throughput, low-cost approach to spatial transcriptomic profiling. A uniform barcoded microarray is crucial for spatially unbiased mRNA analysis. However, non-specific adsorption of barcoding reagents in microchannels occurs during liquid transport, causing nonuniform barcoding in chip functional regions. The uneven microarray further leads to biased transcriptome capture. Herein, we develop a barcode specific immobilization (BarSI) interface with both anti-adsorption property and biological activity for the development of uniform spatially barcoded microarray chips. We immobilize DNA probes in straight and serpentine microchannels with coefficient of variation (CV) of 2.3% and 3.2%. Based on the orthogonal barcoding system, we develop spatially barcoded microarray chips with an overall fluorescence intensity CV of 8.47 ± 1.26%, compared with the CV of 20.91 ± 2.84% of microarrays developed on conventional amino glass slides. Using the uniform spatially barcoded microarray chip, we achieve spatially unbiased detection of mouse liver mRNA with the absolute value of Moran’s I below 0.05. We present an economical and accessible method for manufacturing uniform spatially barcoded microarray chips, introducing a novel strategy for unbiased transcriptome analysis.