Analysis of promoter strength and specificity is important for understanding and engineering gene regulation. Here, we report an in vitro promoter analysis method that can achieve both massiveness and quantitativeness. In this approach, a pool of single-stranded DNA with a partially randomized promoter sequence to be analyzed is chemically synthesized. Through enzymatic reactions, the randomized sequence will be copied to the downstream region, resulting in a template DNA pool that carries its own promoter information on its transcribed region. After in vitro transcription of the DNA pool with an RNA polymerase of interest, the sequences of the resulting transcripts will be analyzed. Since the promoter strength linearly correlates to the copy number of transcript, the strength of each promoter sequence can be evaluated. A model experiment of T7 promoter variants demonstrated the quantitativeness of the method, and the method was applied for the analysis of the promoter of cyanophage Syn5 RNA polymerase. This method provides a powerful approach for analyzing the complexity of promoter specificity and discrimination for highly abundant and often redundant alternative sigma factors such as the extracellular function (ECF) sigma factors.