Background Long non-coding RNAs (lncRNAs) are promising diagnostic and prognostic biomarkers in cancers. Neoadjuvant chemoradiotherapy (NCRT) is the standard of care for patients with locally advanced rectal cancer (LARC). However, studies are limited regarding lncRNAs associated NCRT response and prognosis of LARC patients. This study aimed to identify lncRNAs associated with NCRT response and prognosis in CRC patients, and to explore potential mechanisms. Methods LncRNA expression profiles from our previous gene chip data basing on the LASSO to identify a four-lncRNA signature that predicted NCRT response and prognosis and further validated in 138 colorectal cancer (CRC) patients and 36 LARC patients from our center. A Cox regression model was performed to identify prognostic risk factors. Moreover, we identified the function of the LINC00909 in vivo and in vitro in CRC cell lines. Results Four hub lncRNAs (DBET, LINC00909, FLJ33534, and HSD52) were screened by comparing the relative lncRNA expression of NCRT-responsive and non-responsive patients (AUC = 0.68, 0.73, 0.73, and 0.70, respectively, all p < 0.05). A competing endogenous RNA (ceRNA) network was constructed based on the four lncRNAs. Moreover, the four lncRNAs expression was identified by the external data in cancerous and adjacent non-cancerous tissues in CRC patients. The results demonstrated that the expression of the four lncRNAs was lower in the normal tissues than in the cancerous tissues (all p < 0.05), and the COX analysis demonstrated that the DBET, LINC00909 and FLJ33534 were assocaited with the DFS in CRC patients. The four lncRNAs were also identified in the LARC following NCRT patients, and the result demonstrated that LINC00909 and FLJ33534 had powerful ability to predict the NCRT response and prognosis (all p < 0.05). Basing on the multivariate COX analysis, we constructed a risk score and verified in the CRC and LARC patients in predicting NCRT response and prognosis. Moreover, The expression and prognosis of the DBET, LINC00909 and FLJ33534 in the CRC tissues were identified in the R2 platform and Oncomine database. Moreover, the over-expression LINC00909 cell lines demonstrated that over-expressed the LINC00909 increased the cell lines resistance to the 5-FU and radiotherapy in vivo and in vitro. Conclusion Our findings showed that DBET, LINC00909, and FLJ33534 could serve as novel biomarkers for prediction of NCRT response and prognosis in CRC patients. And LINC00909 could be a novel therapeutic targets in enhancing the NCRT response.