The invention relates to a self-set concentration gradient drug screening organ chip and a preparation method thereof. The organ chip comprises a multi-cell co-culture system, a seed cell culture system, a blank control system and a drug testing system. The organ chip simultaneously can carry out activity or toxicity test and blank control experiment on drugs of eight drug concentration gradients at a time, so that the organ chip is simple in operation, can realize the parallel implantation and co-culture of various cells, reduce the application amount of actual samples, implement low drug consumption, and simplify a cell implantation process, therefore, the organ chip has portable, economical, efficient and accurate characteristics. The organ chip can independently carry out cell growth and culture and carry out multi-concentration gradient detection on drugs, and can carry out in-vitro, real-time and online observation on the biological behaviors of cells, tissues and organs, thereby providing a brand-new technical platform for cell-drug research and high-throughput drug screening.