Background: Spinal muscular atrophy (SMA) is caused by homozygous deletion or compound heterozygous mutation of survival motor neuron gene 1 (SMN1), which is the key to diagnose SMA. The study was to establish and evaluate a new diagnostic method for SMA. Methods: A total of 1494 children suspected with SMA were enrolled in this study. Traditional strategy, including multiplexed ligation-dependent probe amplification (MLPA) and TA cloning, was used in 1364 suspected SMA children from 2003 to 2014, and the 130 suspected SMA children were tested by a new strategy from 2015 to 2016, who were also verified by MLPA combined with TA cloning. The SMN1 and SMN2 were simultaneously amplified by polymerase chain reaction using the same primers. Mutation Surveyor software was used to detect and quantify the SMN1 variants by calculating allelic proportions in Sanger sequencing. Finally, taround time and cost of these two strategies were compared. Results: Among 1364 suspected SMA children, 576 children had SMN1 homozygous deletion and 27 children had SMN1 compound heterozygous mutation. Among the 130 cases, 59 had SMN1 homozygous deletion and 8 had heterozygous deletion: the SMN1-specific peak proportion on exon 7 was 34.6 ± 1.0% and 25.5 ± 0.5%, representing SMN1:SMN2 to be 1:2 and 1:3, respectively. Moreover, five variations, including p.Ser8Lysfs *23 (in two cases), p.Leu228*, p.Pro218Hisfs *26, p.Ser143Phefs*5, and p.Tyr276His, were detected in 6/8 cases with heterozygous deletion, the mutant allele proportion was 31.9%, 23.9%, 37.6%, 32.8%, 24.5%, and 23.6%, which was similar to that of the SMN1-specific site on exon 7, suggesting that those subtle mutations were located in SMN1. All these results were consistent with MLPA and TA cloning. The taround times of two strategies were 7.5 h and 266.5 h, respectively. Cost of a new strategy was only 28.5% of the traditional strategy. Conclusion: Sanger sequencing combined with Mutation Surveyor analysis has potential application in SMA diagnosis.