A display substrate, a manufacturing method thereof, and a display device are provided. The display substrate includes multiple pixel units, multiple scan lines, and multiple data lines. Each pixel unit includes a first sub-pixel, a second sub-pixel, and a third sub-pixel having different colors. Sub-pixels having a same color are in a same sub-pixel row. The data line includes a first sub-data line and a second sub-data line, the first sub-pixels on two sides of each first sub-data line are electrically connected to a same first sub-data line and are driven through a same first sub-data line, and the second sub-pixel and the third sub-pixel in a same column are electrically connected to a same second sub-data line and are driven through a same second sub-data line.