To develop a molecular probe for MRI detection of human tumor telomerase reverse transcriptase (hTERT) mRNA expression. Uniformly phosphorothioate‐modified hTERT antisense oligonucleotide (ASON) homing hTERT mRNA was labeled with gadolinium (Gd) through the bifunctional chelator 1,4,7, 10‐tetraazacyclododecane‐N, N', N'', N'''‐tetraacetic acid (DOTA) stirred within 45 minutes at 60°C. The Gd labeled probes were characterized in vitro. The cellular uptake rate and biodistribution of 99mTc‐DOTA‐ASON was measured instead of that of Gd‐DOTA‐ASON. A549 lung adenocarcinoma model was established in BALB/c nude mice and Gd‐DOTA‐ASON was injected intraperitoneally and MR images were acquired using 7.0T Micro‐MRI (Bruker Biospec, Ettlingen, Germany) at different time points. Immunohistochemical analysis of telomerase activity of each xenograft was operated two days after in vivo imaging. The binding efficiency of Gd‐DOTA‐ASON reached as high as 71.7 ± 4.5% (n = 6). Gd‐DOTA‐ASON displayed perfect stability in fresh human serum at 37°C for 24 h. Compared with normal lung cells, A549 cells showed an obviously higher uptake of 99mTc‐DOTA‐ASON than that of lung cells (10.5 ± 2.7% vs. 4.8 ± 2.6%, P < 0.05). The signal intensity of A549 xenografts can be enhanced by Gd‐DOTA‐ASON and the signal to noise ratio (SNR) of tumor to muscle reached 2.37 and maintained a relatively high level within 6 h after injection. The activity of hTERT in A549 tumors can be suppressed by Gd‐DOTA‐ASON in pathological slices. The results of this study show that Gd‐DOTA‐ASON can be a promising intracellular MR contrast probe for targeting telomerase‐positive carcinomas. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02316.x, 2012)