Western blot (WB) is widely used to investigate the expression profiling for target proteins，which depends on the specific binding with antibodies and it is a fundamental technique in basic and application field of life science. However, traditional WB technique involves many manual steps, it is difficult to set up standard operational protocols. In most of the cases, it is used only within same WB analysis for the abundance investigation of target proteins qualitatively or relative quantitatively, while problematic to carry out comparison among different laboratories. In the current paper, after a summarized historical review of WB development, the concept of advance version of WB (WB 2.0) was proposed. The key components for the design and practical steps including digitalization, standardization, automation, micro-quantification, high through-put, reference-based normalization and database establishment were presented. In perspective, the application of WB 2.0 will activate the establishment of a public accessible protein expression database, which will be another supporting platform for life science succeeding the recognized genome and transcriptome databases.