DNA sequencing technology has markedly advanced the development of biological and medicinal sciences. High-throughput pyrosequencing instruments that combine the pyrosequencing with microfabricated high-density picoliter reactors have been proved to be suitable for de novo sequencing and metagenome sequencing. In the present work, we report on an alternative sequencing chip consisting of hundreds of thousands of picoliter sized honeycombed SU8 reaction vessels on a fiber-optic slide by lithography technique for high-throughput pyrosequencing instruments. Highly reproducible fabrication process of SU8 sequencing chip is achieved through the improvement on SU8 film thickness uniformity and relaxation of SU8 residual stress during fabrication. To achieve the optical isolation required for SU8 reaction wells, metal film is selectively deposited on the side walls of the reaction vessels by reformating vacuum coating. With the metal coating, the average value of optical cross talking between SU8 reaction vessels is reduced from 25% to 1%. The SU8 sequencing chip demonstrates an excellent light transmission characteristic and meets the need of pyrosequencing application.