The potential mechanism of Linggui Zhugan Decoction (LZD) on myocardial fibrosis (MF) after acute myocardial infarction (AMI) was investigated by bioinformatics and in vivo. The active component of LZD and the corresponding target persons were calculated from the TCMSP database. Collecting disease targets in GeneCards, OMIM, TTD and GEO databases; Mapping the drug-ingredient-target network using Cytoscape software; STRING website construction protein-protein interaction network; Enrichment analysis for R language. The MF model of AMI was developed after left anterior descending coronary artery elevation in mice.. Four weeks later, the cardiac function of the mice was detected by Doppler ultrasound imager. Changes in heart shape and collagen fiber deposition were observed by HE and Masson staining. Myocardial fibrosis and core target protein expression were detected by Western blot. The level of α-smooth muscle actin (α-SMA) in myocardial tissue was detected by immunohistochemistry. The bioinformatics results showed that there were 33 intersection targets of the active components of LZD, disease targets and GSE97358 differential genes. The core active ingredients are quercetin, kaempferol, β-sitosterol, etc. The results of molecular docking showed that the core target had good docking with the main active components of LZD. The experimental results showed that LZD could significantly improve the cardiac function and pathologic changes of MF mice after AMI, and the interstitial collagen fiber deposition was significantly reduced. The expression levels of fibrosis-related proteins α-SMA, collagen Ⅰ (Col Ⅰ), Col Ⅲ, matrix metalloproteinase-2 (MMP-2) and core targets interleukin-1β (IL-1β), prostaglandin-endoperoxide synthase 2 (PTGS2) and CXC chemokine ligand 8 (CXCL8) in mouse myocardial tissue were significantly decreased, while the expression levels of peroxisome proliferators activated receptor gamma (PPARγ) and B-cell lymphoma-2 (Bcl-2) were significantly increased. In summary, LZD can regulate multiple signaling pathways and targets, significantly improve MF after AMI in mice, and delay the pathological process of AMI transformation into chronic heart failure. The mechanism of action may be closely related to the target obtained by bioinformatics screening.