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Simple Summary: High light stress is one of the main factors affecting the normal growth of Ulva prolifera. The response mechanism of U. prolifera to 12 h of high light stress was explored by the multi-omics method. We found that short-term high light could inhibit the assimilation process of U. prolifera, destroy the cellular structure, and inhibit respiration. Moreover, it was raised by the genes associated with photosynthetic pigment synthesis, optical system I, and electronic transport, and may be able to make up the ATP defects by circulating electronic transport. At the same time, it reduced NADPH production by attenuating photosystem II synthesis. The carbon fixed approach was also transformed from the C3 pathway to the C4 pathway. Revealing the response mechanism of U. prolifera to high light can provide a more theoretical basis for studying the outbreak of green tide of U. prolifera in the Yellow Sea. The main algal species of Ulva prolifera green tide in the coastal areas of China are four species, but after reaching the coast of Qingdao, U. prolifera becomes the dominant species, where the light intensity is one of the most important influencing factors. In order to explore the effects of short-term high light stress on the internal molecular level of cells and its coping mechanism, the transcriptome, proteome, metabolome, and lipid data of U. prolifera were collected. The algae were cultivated in high light environment conditions (400 μmol·m−2·s−1) for 12 h and measured, and the data with greater relative difference (p < 0.05) were selected, then analyzed with the KEGG pathway. The results showed that the high light stress inhibited the assimilation of U. prolifera, destroyed the cell structure, and arrested its growth and development. Cells entered the emergency defense state, the TCA cycle was weakened, and the energy consumption processes such as DNA activation, RNA transcription, protein synthesis and degradation, and lipid alienation were inhibited. A gradual increase in the proportion of the C4 pathway was recorded. This study showed that U. prolifera can reduce the reactive oxygen species produced by high light stress, inhibit respiration, and reduce the generation of NADPH. At the same time, the C3 pathway began to change to the C4 pathway which consumed more energy. Moreover, this research provides the basis for the study of algae coping with high light stress. [ABSTRACT FROM AUTHOR]